Antifungal polypeptides

ABSTRACT

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/585,267, filed on Jul. 2, 2004, which is herein incorporated byreference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contract numberDE-AC02-05CH11231 awarded by the United States Department of Energy. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to polypeptides having antipathogenicactivity and the nucleic acid sequences that encode them. Methods of theinvention utilize these antipathogenic polypeptides and nucleic acidsequences to control plant pathogens and to increase pathogen resistancein plants.

BACKGROUND OF THE INVENTION

Plant diseases are often a serious limitation on agriculturalproductivity and therefore have influenced the history and developmentof agricultural practices. A variety of pathogens are responsible forplant diseases, including fungi, bacteria, viruses, and nematodes. Amongthe causal agents of infectious diseases of crop plants, however, fungiare the most economically important group of plant pathogens and areresponsible for huge annual losses of marketable food, fiber, and feed.

Incidence of plant diseases has traditionally been controlled byagronomic practices that include crop rotation, the use ofagrochemicals, and conventional breeding techniques. The use ofchemicals to control plant pathogens, however, increases costs tofarmers and causes harmful effects on the ecosystem. Consumers andgovernment regulators alike are becoming increasingly concerned with theenvironmental hazards associated with the production and use ofsynthetic agrochemicals for protecting plants from pathogens. Because ofsuch concerns, regulators have banned or limited the use of some of themost hazardous chemicals. The incidence of fungal diseases has beencontrolled to some extent by breeding resistant crops. Traditionalbreeding methods, however, are time-consuming and require continuouseffort to maintain disease resistance as pathogens evolve. See, forexample, Grover and Gowthaman (2003) Curr. Sci. 84:330-340. Thus, thereis a significant need for novel alternatives for the control of plantpathogens that possess a lower risk of pollution and environmentalhazards than is characteristic of traditional agrochemical-based methodsand that are less cumbersome than conventional breeding techniques.

Many plant diseases, including, but not limited to, maize stalk rot andear mold, can be caused by a variety of pathogens. Stalk rot, forexample, is one of the most destructive and widespread diseases ofmaize. The disease is caused by a complex of fungi and bacteria thatattack and degrade stalks near plant maturity. Significant yield losscan occur as a result of lodging of weakened stalks as well as prematureplant death. Maize stalk rot is typically caused by more than one fungalspecies, but Gibberella stalk rot, caused by Gibberella zeae, Fusariumstalk rot, caused by Fusarium verticillioides, F. proliferatum, or F.subglutinans, and Anthracnose stalk rot, caused by Colletotrichumgraminicola are the most frequently reported (Smith and White (1988);Diseases of corn, pp. 701-766 in Corn and Corn Improvement, AgronomySeries #18 (3rd ed.), Sprague, C. F., and Dudley, J. W., eds. Madison,Wis.). Due to the fact that plant diseases can be caused by a complex ofpathogens, broad spectrum resistance is required to effectively mediatedisease control. Thus, a significant need exists for antifungalcompositions that target multiple stalk rot and ear mold-causingpathogens.

Recently, agricultural scientists have developed crop plants withenhanced pathogen resistance by genetically engineering plants toexpress antipathogenic proteins. For example, potatoes and tobaccoplants genetically engineered to produce an antifungal endochitinaseprotein were shown to exhibit increased resistance to foliar andsoil-borne fungal pathogens. See Lorito et al. (1998) Proc. Natl. Acad.Sci. 95:7860-7865.

Moreover, transgenic barley that is resistant to the stem rust fungushas also been developed. See Horvath et al. (2003) Proc. Natl. Acad.Sci. 100:364-369. A continuing effort to identify antipathogenic agentsand to genetically engineer disease-resistant plants is underway.

Thus, in light of the significant impact of plant pathogens,particularly fungal pathogens, on the yield and quality of crops, newcompositions and methods for protecting plants from pathogens areneeded. Methods and compositions for controlling multiple fungalpathogens are of particular interest.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for protecting a plant from a pathogen areprovided. The compositions include novel nucleotide and amino acidsequences for antipathogenic, particularly antifungal, polypeptides. Thepolypeptides of the invention display antipathogenic activity againstplant fungal pathogens. More particularly, the compositions of theinvention comprise the antipathogenic polypeptides set forth in SEQ IDNOs:1, 3, 5, 7, and 9, and variants and fragments thereof. Nucleic acidmolecules comprising nucleotide sequences that encode the antipathogenicpolypeptides of the invention are further provided. Compositions alsoinclude expression cassettes comprising a promoter operably linked to anucleotide sequence that encodes an antipathogenic polypeptide of theinvention. Transformed plants, plant cells, seeds, and microorganismscomprising an expression cassette of the invention are further provided.

The compositions of the invention are useful in methods directed toinducing pathogen resistance, particularly fungal resistance, in plants.In particular embodiments, the methods comprise introducing into a plantat least one expression cassette comprising a promoter operably linkedto a nucleotide sequence that encodes an antipathogenic polypeptide ofthe invention. As a result, the antipathogenic polypeptide is expressedin the plant, and the pathogen is exposed to the protein at the site ofpathogen attack, thereby leading to increased pathogen resistance. Atissue-preferred promoter may be used to drive expression of anantipathogenic protein in specific plant tissues that are particularlyvulnerable to pathogen attack, such as, for example, the roots, leaves,stalks, vascular tissues, and seeds. Pathogen-inducible promoters mayalso be used to drive expression of an antipathogenic protein of theinvention at or near the site of pathogen infection.

The present invention further provides antipathogenic compositions andformulations and methods for their use in protecting a plant from apathogen, particularly a fungal pathogen. In some embodiments,compositions comprise an antipathogenic polypeptide or a transformedmicroorganism comprising a nucleotide sequence encoding anantipathogenic polypeptide of the invention in combination with acarrier. Methods of using these compositions to protect a plant from apathogen comprise applying the antipathogenic composition to theenvironment of the plant pathogen by, for example, spraying, dusting,broadcasting, or seed coating. The methods and compositions of theinvention find use in protecting plants from pathogens, including fungalpathogens, viruses, nematodes, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence alignment of the amino acid sequences of theinvention with various known proteins sharing sequence similarity to thepolypeptides disclosed herein. Consensus sequences derived from thehighlighted region of all polypeptides appearing in the alignment arefurther provided.

FIG. 2 shows a sequence alignment of the novel amino acid sequences ofthe invention.

FIG. 3 shows photographic examples of the level of inhibition associatedwith each numerical score in the antifungal plate assay described inExample 3.

FIG. 4 provides the photographic results of antifungal activity assaysperformed with the polypeptide set forth in SEQ ID NO:1, as described inExample 3. Antifungal activity against Colletotrichum graminicola,Diplodia maydis, Fusarium graminearum, and Fusarium verticillioides wasobserved.

FIGS. 5A and 5B show the results of antifungal activity assays performedwith the polypeptides set forth in SEQ ID NO:3 and SEQ ID NO:9,respectively. Both polypeptides inhibited Colletotrichum graminicola,Diplodia maydis, Fusarium graminearum, and Fusarium verticillioides in adose-dependent fashion. Experimental details are provided in Example 3.

FIG. 6 provides the results of a Fusarium verticillioides challengeassay of maize seedlings expressing the antifingal polypeptidedesignated LBNL-5220 (SEQ ID NO:1). Experimental details are provided inExample 8. Results obtained with negative control seedlings transformedwith an empty vector control plasmid are presented as a hatched bar.White bars represent results from maize seedlings transformed with theLBNL-5220 construct in which a statistically significant difference (atthe 95% confidence level) in GUS activity relative to that of controlsamples was observed. Transformation events in which decreases in GUSactivity relative to controls were not statistically significant (at the95% confidence level) are presented as black bars.

FIG. 7 provides the photographic results of a F. verticillioidesantifungal activity assay performed using transgenic LBNL-5220 (SEQ IDNO:1) callus extracts. Experimental details are provided in Example 9.

FIG. 8 provides the results of Colletotrichum graminicola resistanceassays of transgenic LBNL-5220 (SEQ ID NO:1) maize plants. Experimentaldetails are provided in Example 10. Results obtained with negativecontrols (i.e., empty vector control events) are presented as a hatchedbar. Results obtained in upper stalk internodes of transformation eventsmarked with white bars were statistically more resistant to infection byC. graminicola than negative control stalks. Transformation events inwhich a statistically significant improvement in fungal resistance wasnot observed are presented as black bars.

FIG. 9 provides the distribution of upper stalk C. graminicolaresistance scores for control and LBNL-5220 transgenic maize plants.Experimental details are provided in Example 10.

FIG. 10 provides the distribution of lower stalk C. graminicolaresistance scores for control and LBNL-5220 transgenic maize plants.Experimental details are provided in Example 10.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods directed toinducing pathogen resistance, particularly fungal resistance, in plants.The compositions are novel nucleotide and amino acid sequences forantipathogenic polypeptides. Specifically, the present inventionprovides antipathogenic polypeptides having the amino acid sequences setforth in SEQ ID NOs:1, 3, 5, 7, and 9, and variants and fragmentsthereof, that were isolated from various microbial fermentation broths.Isolated nucleic acid molecules, and variants and fragments thereof,comprising nucleotide sequences that encode the amino acid sequencesshown in SEQ ID NOs:1, 3, 5, 7, and 9 are further provided.

Nucleotide sequences that are optimized for expression in plants,particularly maize, and that encode the polypeptides of SEQ ID NOs:1, 3,5, 7, and 9 were generated using standard methods known in the art.These deduced nucleotide sequences are set forth in SEQ ID NOs:2, 4, 6,8, and 10. Plants, plant cells, seeds, and microorganisms comprising anucleotide sequence that encodes an antipathogenic polypeptide of theinvention are also disclosed herein. Antipathogenic compositionscomprising an isolated antipathogenic, particularly an antifungal,polypeptide or a microorganism that expresses a polypeptide of theinvention in combination with a carrier are further provided. Thecompositions of the invention find use in generating pathogen-resistantplants and in protecting plants from pathogens, particularly fungalpathogens.

The polypeptides disclosed herein in SEQ ID NOs:1, 3, 5, 7, and 9display antifungal activity against fungal plant pathogens, such as, forexample, Colletotrichum graminocola, Diplodia maydis, Fusariumgraminearum, and Fusarium verticillioides. The species of origin ofthese antifungal polypeptides has been determined. These proteins are offilamentous fungal origin. In particular, the fungal source ofpolypeptide SEQ ID NO:1 is Penicillium simplicissimum, while the fungalsource of the polypeptides SEQ ID NOs:3 and 5 are two strains of thespecies Penicillium miczyinskii. The fungal source of the polypeptidesof SEQ ID NOs:7 and 9 is Monascus ruber.

Additional novel polypeptides are provided that share homology with theamino acid sequences set forth in SEQ ID NOs:1, 3, 5, 7, and 9. Inparticular, the polypeptides set forth in SEQ ID NOs:11 and 15 wereidentified using computer homology searches from a fungal contaminant ofmaize and from Fusarium graminearum, respectively. Mature peptideslacking an N-terminal peptide present in the full-length sequences ofSEQ ID NO:11 and 15 are also disclosed and set forth in SEQ ID NOs: 13and 17. Isolated nucleic acid molecules, and variants and fragmentsthereof, comprising nucleotide sequences that encode the amino acidsequences shown in SEQ ID NOs:11, 13, 15, and 17 are further provided.Nucleotide sequences that encode these polypeptides have been generated,and these deduced sequences are set forth in SEQ ID NOs:12, 14, 16, and18.

The polypeptides of the invention share homology with known antifungalproteins, as well as other proteins of unknown function. In particular,the novel polypeptides of the invention share homology with antifungalproteins isolated from Aspergillus giganteus (Accession No. AAE78772;SEQ ID NO:59) and Penicillium chrysogenum (Accession No. AAC86132; SEQID NO:60) and a protein of unknown function isolated from Aspergillusniger (Accession No. CAD66625; SEQ ID NO:61). See U.S. Pat. Nos.5,747,285, 5,804,184, and 6,271,438 and International Publication No. WO02/09034, herein incorporated by reference in their entirety. FIG. 1provides an alignment of the amino acid sequences set forth in SEQ IDNOs:1, 3, 5, 7, 9, 13, and 17 with the A. giganteus and P. chrysogenumantifungal proteins and the A. niger protein of unknown function.

The novel amino acid sequences disclosed comprise the consensus sequenceshown in SEQ ID NO:68 (i.e.,G-X-C-X-X-X-X-N-X-C-X-Y-X-X-X-X-X-X-X-X-Z-V-X-C-X-X-X-A-N-X-X-C-X-X-D-X-X-X-C-X-X-D-X-X-X-X-X-V-X-C,wherein “X” refers to any one amino acid and “Z” refers to any one aminoacid or no amino acid.) The consensus sequences may be more specificallyrepresented by the sequences set forth in SEQ ID NO:39(G-X-C-X-X-X-X-N-X-C-X-Y-X-X-X-X-X-X-X-X-V-X-C-X-X-X-A-N-X-X-C-X-X-D-X-X-X-C-X-X-D-X-X-X-X-X-V-X-C)or SEQ ID NO:40(G-X-C-X-X-X-X-N-X-C-X-Y-X-X-X-X-X-X-X-X-X-V-X-C-X-X-X-A-N-X-X-C-X-X-D-X-X-X-C-X-X-D-X-X-X-X-X-V-X-C),wherein “X” refers to any amino acid. This consensus sequence is uniqueamong the known antifungal proteins. The consensus sequence disclosedherein may further comprise the consensus sequence as shown in SEQ IDNO:41 ((K or Q)-(Y or F)-X-G-X-C-X-X-X-X-N-X-C-(T orK)-Y-X-X-X-X-X-X-X-X-V-X-C-(P or G)-(S or T)-(A or F)-A-N-X-(R orK)-C-X-X-D-(R or G)-X-X-C-X-(Y or F)-D-X-(H or Y)-X-X-X-V-X-C-(Q or D))or SEQ ID NO:42: ((K or Q)-(Y or F)-X-G-X-C-X-X-X-X-N-X-C-(T orK)-Y-X-X-X-X-X-X-X-X-X-V-X-C-(P or G)-(S or T)-(A or F)-A-N-X-(R orK)-C-X-X-D-(R or G)-X-X-C-X-(Y or F)-D-X-(H or Y)-X-X-X-V-X-C-(Q or D)),wherein “X” refers to any amino acid, and wherein “or” indicates one orthe other amino acids (of the two indicated in parentheses) may besubstituted at the respective positions.

Alignment data indicate that SEQ ID NO:1 shares approximately 49%sequence identity with the A. giganteus antifungal protein and 58%sequence identity with the P. chrysogenum antifungal protein. SEQ IDNOs:5 and 7 share approximately 81% and 79% sequence identity with theA. niger protein of unknown function, respectively. Overall, thepolypeptide sequences of the invention share about 26-45% sequenceidentity with the A. giganteus antifungal protein, about 25-60% sequenceidentity with the P. chrysogenum antifungal protein, and about 26-86%sequence identity with the A. niger protein. Tables summarizing theglobal identity and similarity data are provided in Table 1A and 1B.

The A. giganteus antifungal protein causes membrane permeabilization ofsusceptible fungi and exhibits potent antifungal activity against thephytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme andthe oomycete pathogen Phytophthora infestans. See, for example, Vila etal. (2001) Mol. Plant. Microbe Interact. 14:1327-31; Theis et al. (2003)Antimicrob. Agents Chemother. 47:588-93; and Meyer et al. (2003) J.Basic Microbiol. 43:68-74. Moreover, wheat transformed with the A.giganteus antifungal protein shows increased fungal resistance toErysiphe graminis and Puccinia recondite. See, for example, Oldach etal. (2001) Mol. Plant Microbe Interact. 14:832-8. Similarly, theantifungal protein isolated from P. chrysogenum has been shown to causemembrane permeabilization and ion leakage in certain fungi and toinhibit the growth of various filamentous fungi. See, for example,Kaiserer et al. (2003) Arch. Microbiol. 180:204-10.

The nucleic acids and polypeptides of the present invention find use inmethods for inducing pathogen resistance in a plant. Accordingly, thecompositions and methods disclosed herein are useful in protectingplants against fungal pathogens, viruses, nematodes and the like.“Pathogen resistance” or “disease resistance” is intended to mean thatthe plant avoids the disease symptoms that are the outcome ofplant-pathogen interactions. That is, pathogens are prevented fromcausing plant diseases and the associated disease symptoms, oralternatively, the disease symptoms caused by the pathogen are minimizedor lessened, such as, for example, the reduction of stress andassociated yield loss. One of skill in the art will appreciate that thecompositions and methods disclosed herein can be used with othercompositions and methods available in the art for protecting plants frominsect and pathogen attack.

“Antipathogenic compositions” or “antipathogenic polypeptides” isintended to mean that the compositions of the invention haveantipathogenic activity and thus are capable of suppressing,controlling, and/or killing the invading pathogenic organism. Anantipathogenic polypeptide of the invention will reduce the diseasesymptoms resulting from pathogen challenge by at least about 5% to about50%, at least about 10% to about 60%, at least about 30% to about 70%,at least about 40% to about 80%, or at least about 50% to about 90% orgreater. Hence, the methods of the invention can be utilized to protectplants from disease, particularly those diseases that are caused byplant pathogens. In particular embodiments, the antipathogenic activityexhibited by the polypeptides of the invention is antifungal activity.As used herein, “antifingal activity” refers to the ability to suppress,control, and/or kill the invading fungal pathogen. Likewise, “fungalresistance” refers to enhanced tolerance to a fungal pathogen whencompared to that of an untreated or wild type plant. Resistance may varyfrom a slight increase in tolerance to the effects of the fungalpathogen (e.g., partial inhibition) to total resistance such that theplant is unaffected by the presence of the fungal pathogen. An increasedlevel of resistance against a particular fungal pathogen or against awider spectrum of fungal pathogens may both constitute antifungalactivity or improved fungal resistance.

Assays that measure antipathogenic activity are commonly known in theart, as are methods to quantitate disease resistance in plants followingpathogen infection. See, for example, U.S. Pat. No. 5,614,395, hereinincorporated by reference. Such techniques include, measuring over time,the average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. For example, a plant eitherexpressing an antipathogenic polypeptide or having an antipathogeniccomposition applied to its surface shows a decrease in tissue necrosis(i.e., lesion diameter) or a decrease in plant death following pathogenchallenge when compared to a control plant that was not exposed to theantipathogenic composition. Alternatively, antipathogenic activity canbe measured by a decrease in pathogen biomass. For example, a plantexpressing an antipathogenic polypeptide or exposed to an antipathogeniccomposition is challenged with a pathogen of interest. Over time, tissuesamples from the pathogen-inoculated tissues are obtained and RNA isextracted. The percent of a specific pathogen RNA transcript relative tothe level of a plant specific transcript allows the level of pathogenbiomass to be determined. See, for example, Thomma et al. (1998) PlantBiology 95:15107-15111, herein incorporated by reference.

Furthermore, in vitro antipathogenic assays include, for example, theaddition of varying concentrations of the antipathogenic composition topaper disks and placing the disks on agar containing a suspension of thepathogen of interest. Following incubation, clear inhibition zonesdevelop around the discs that contain an effective concentration of theantipathogenic polypeptide (Liu et al. (1994) Plant Biology91:1888-1892, herein incorporated by reference). Additionally,microspectrophotometrical analysis can be used to measure the in vitroantipathogenic properties of a composition (Hu et al. (1997) Plant Mol.Biol. 34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233,both of which are herein incorporated by reference). Assays thatspecifically measure antifungal activity are also well known in the art.See, for example, Duvick et al. (1992) J. Biol. Chem. 267:18814-18820;Lacadena et al. (1995) Arch. Biochem. Biophys. 324:273-281; Xu et al.(1997) Plant Mol. Biol. 34: 949-959; Lee et al. (1999) Biochem. Biophys.Res. Comm. 263:646-651; Vila et al. (2001) Mol. Plant Microbe Interact.14:1327-1331; Moreno et al. (2003) Phytpathol. 93:1344-1353; Kaiserer etal. (2003) Arch. Microbiol. 180:204-210; and U.S. Pat. No. 6,015,941.

The compositions disclosed herein comprise isolated nucleic acids thatencode antipathogenic polypeptides, expression cassettes comprising thenucleotide sequences of the invention, and isolated antipathogenicpolypeptides. Antipathogenic compositions comprising a polypeptide ofthe invention in combination with a carrier are also provided. Theinvention further discloses plants and microorganisms transformed withnucleic acids that encode antipathogenic proteins. The compositions finduse in methods for inducing pathogen resistance in a plant and forprotecting a plant from a pathogen, particularly fungal pathogens.

In particular aspects, methods for inducing pathogen resistance in aplant comprise introducing into a plant at least one expressioncassette, wherein the expression cassette comprises a nucleotidesequence encoding an antipathogenic polypeptide of the inventionoperably linked to a promoter that drives expression in the plant. Theplant expresses the antipathogenic polypeptide, thereby exposing thepathogen to the polypeptide at the site of pathogen attack. Inparticular embodiments, the polypeptide has antifungal activity, and thepathogen is a fungus, such as, for example, Colletotrichum graminicola,Diplodia maydis, Fusarium graminearum, or Fusarium verticillioides.Expression of an antipathogenic polypeptide of the invention may betargeted to specific plant tissues where pathogen resistance isparticularly important, such as, for example, the leaves, roots, stalks,or vascular tissues. Such tissue-preferred expression may beaccomplished by root-preferred, leaf-preferred, vasculartissue-preferred, stalk-preferred, or seed-preferred promoters.Moreover, the polypeptides of the invention may also be targeted tospecific subcellular locations within a plant cell or, alternatively,secreted from the cell, as described herein below.

Just as expression of an antipathogenic polypeptide of the invention maybe targeted to specific plant tissues or cell types through the use ofappropriate promoters, it may also be targeted to different locationswithin the cell through the use of targeting information or “targetinglabels.” Unlike the promoter, which acts at the transcriptional level,such targeting information is part of the initial translation product.Depending on the mode of infection of the pathogen or the metabolicfunction of the tissue or cell type, the location of the protein indifferent compartments of the cell may make it more efficacious againsta given pathogen or make it interfere less with the functions of thecell. For example, one may produce a protein preceded by a signalpeptide, which directs the translation product into the endoplasmicreticulum, by including in the construct (i.e. expression cassette)sequences encoding a signal peptide (such sequences may also be calledthe “signal sequence”). The signal sequence used could be, for example,one associated with the gene encoding the polypeptide, or it may betaken from another gene. There are many signal peptides described in theliterature, and they are largely interchangeable (Raikhel andChrispeels, “Protein sorting and vesicle traffic” in Buchanan et al.,eds, (2000) Biochemistry and Molecular Biology of Plants (AmericanSociety of Plant Physiologists, Rockville, Md.), herein incorporated byreference). The addition of a signal peptide will result in thetranslation product entering the endoplasmic reticulum (in the processof which the signal peptide itself is removed from the polypeptide), butthe final intracellular location of the protein depends on otherfactors, which may be manipulated to result in localization mostappropriate for the pathogen and cell type. The default pathway, thatis, the pathway taken by the polypeptide if no other targeting labelsare included, results in secretion of the polypeptide across the cellmembrane (Raikhel and Chrispeels, supra) into the apoplast. The apoplastis the region outside the plasma membrane system and includes cellwalls, intercellular spaces, and the xylem vessels that form acontinuous, permeable system through which water and solutes may move.This will often be a suitable location. In particular embodiments, anucleotide sequence encoding a barley alpha-amylase signal peptide (SEQID NO:34) is joined in frame with a polynucleotide of the invention.See, for example, the nucleotide sequences set forth in SEQ ID NOs:19,21, 23, 25, 27, 29, and 31.

Other pathogens may be more effectively combated by locating the peptidewithin the cell rather than outside the cell membrane. This can beaccomplished, for example, by adding an endoplasmic reticulum retentionsignal encoding sequence to the sequence of the gene. Methods andsequences for doing this are described in Raikhel and Chrispeels, supra;for example, adding sequences encoding the amino acids K, D, E and L inthat order, or variations thereof described in the literature, to theend of the protein coding portion of the polypeptide will accomplishthis. ER retention sequences are well known in the art and include, forexample, KDEL (SEQ ID NO:62), SEKDEL (SEQ ID NO:63), HDEL (SEQ IDNO:64), and HDEF (SEQ ID NO:65). See, for example, Denecke et al.(1992). EMBO J. 11:2345-2355; Wandelt et al. (1992) Plant J. 2:181-192;Denecke et al. (1993) J. Exp. Bot. 44:213-221; Vitale et al. (1993) J.Exp. Bot. 44:1417-1444; Gomord et al. (1996) Plant Physiol. Biochem.34:165-181; Lehmann et al. (2001) Plant Physiol. 127 (2): 436-449.

Alternatively, the use of vacuolar targeting labels such as thosedescribed by Raikhel and Chrispeels, supra, in addition to a signalpeptide will result in localization of the peptide in a vacuolarstructure. As described in Raikhel and Chrispeels, supra, the vacuolartargeting label may be placed in different positions in the construct.Use of a plastid transit peptide encoding sequence instead of a signalpeptide encoding sequence will result in localization of the polypeptidein the plastid of the cell type chosen (Raikhel and Chrispeels, supra).Such transit peptides are known in the art. See, for example, Von Heijneet al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J.Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481. Chloroplasttargeting sequences that encode such transit peptides are also known inthe art and include the chloroplast small subunit ofribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filhoet al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J.Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphatesynthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb.22(6):789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem.270(11):6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem.272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J.Biol. Chem. 268(36):27447-27457); and the light harvesting chlorophylla/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem.263:14996-14999).

One could also envision localizing the polypeptide in other cellularcompartments by addition of suitable targeting information. (Raikhel andChrispeels, supra). A useful site available on the world wide web thatprovides information and references regarding recognition of the varioustargeting sequences can be found at: psort.nibb.ac.jp/mit. Otherreferences regarding the state of the art of protein targeting includeSilva-Filho (2003) Curr. Opin. Plant Biol. 6:589-595; Nicchitta (2002)Curr. Opin. Cell Biol. 14:412-416; Bruce (2001) Biochim Biophys Acta1541: 2-21; Hadlington & Denecke (2000) Curr. Opin. Plant Biol. 3:461-468; Emanuelsson et al. (2000) J. Mol. Biol. 300: 1005-1016;Emanuelsson & von Heijne (2001) Biochim Biophys Acta 1541: 114-119,herein incorporated by reference.

The compositions of the invention find further use in methods directedto protecting a plant from a pathogen. “Protecting a plant from apathogen” is intended to mean killing the pathogen or preventing orlimiting disease formation on a plant. In some embodiments, anantipathogenic composition comprising an antipathogenic polypeptide anda carrier is applied directly to the environment of a plant pathogen,such as, for example, on a plant or in the soil or other growth mediumsurrounding the roots of the plant, in order to protect the plant frompathogen attack. Transformed microorganisms comprising a nucleotidesequence encoding an antipathogenic protein of the invention and methodsof using them to protect a plant from a pathogen are further provided.In some embodiments, the transformed microorganism is applied directlyto a plant or to the soil in which a plant grows.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. Polypeptides of the invention can be produced either from anucleic acid disclosed herein, or by the use of standard molecularbiology techniques. For example, a truncated protein of the inventioncan be produced by expression of a recombinant nucleic acid of theinvention in an appropriate host cell, or alternatively by a combinationof ex vivo procedures, such as protease digestion and purification.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.“Fragment” is intended to mean a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence haveantipathogenic activity, more particularly antifungal activity.Alternatively, fragments of a nucleotide sequence that are useful ashybridization probes generally do not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding thepolypeptides of the invention.

A fragment of a nucleotide sequence that encodes a biologically activeportion of an antifungal polypeptide of the invention will encode atleast 15, 25, 30, 40, or 50 contiguous amino acids, or up to the totalnumber of amino acids present in a full-length antifungal polypeptide ofthe invention (for example, 55 amino acids for SEQ ID NO:1). Fragmentsof a nucleotide sequence that are useful as hybridization probes or PCRprimers generally need not encode a biologically active portion of anantipathogenic protein.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide means having the entire nucleic acid sequence of a nativesequence. “Native sequence” is intended to mean an endogenous sequence,i.e., a non-engineered sequence found in an organism's genome.

Thus, a fragment of a nucleotide sequence of the invention may encode abiologically active portion of an antipathogenic polypeptide, or it maybe a fragment that can be used as a hybridization probe or PCR primerusing methods disclosed below. A biologically active portion of anantipathogenic polypeptide can be prepared by isolating a portion of oneof the nucleotide sequences of the invention, expressing the encodedportion of the antipathogenic protein (e.g., by recombinant expressionin vitro), and assessing the activity of the encoded portion of theantifungal protein. Nucleic acid molecules that are fragments of anucleotide sequence of the invention comprise at least 15, 20, 50, 75,100, or 150 contiguous nucleotides, or up to the number of nucleotidespresent in a full-length nucleotide sequence disclosed herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. One of skill in the artwill recognize that variants of the nucleic acids of the invention willbe constructed such that the open reading frame is maintained. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the antipathogenic polypeptides of the invention.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant polynucleotides also includesynthetically derived polynucleotide, such as those generated, forexample, by using site-directed mutagenesis but which still encode anantipathogenic protein of the invention. Generally, variants of aparticular polynucleotide of the invention will have at least about 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptides of SEQ ID NOs:1, 3, 5, 7, and 9 are disclosed. Percentsequence identity between any two polypeptides can be calculated usingsequence alignment programs and parameters described elsewhere herein.Where any given pair of polynucleotides of the invention is evaluated bycomparison of the percent sequence identity shared by the twopolypeptides they encode, the percent sequence identity between the twoencoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, antipathogenic, particularly antifungal,activity as described herein. Such variants may result from, forexample, genetic polymorphism or from human manipulation. Biologicallyactive variants of a native antipathogenic protein of the invention willhave at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the amino acid sequence for the native protein as determinedby sequence alignment programs and parameters described elsewhereherein. A biologically active variant of a protein of the invention maydiffer from that protein by as few as 1-15 amino acid residues, as fewas 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 aminoacid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of theantipathogenic proteins can be prepared by mutations in the DNA. Methodsfor mutagenesis and polynucleotide alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be optimal.

Thus, the genes and polynucleotides of the invention include both thenaturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass naturally occurring proteins as wellas variations and modified forms thereof. Such variants will continue topossess the desired antipathogenic, particularly antifungal, activity.Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and optimallywill not create complementary regions that could produce secondary mRNAstructure. See, EP Patent No. 0075444.

In nature, some polypeptides are produced as complex precursors which,in addition to targeting labels such as the signal peptides discussedelsewhere in this application, also contain other fragments of peptideswhich are removed (processed) at some point during protein maturation,resulting in a mature form of the polypeptide that is different from theprimary translation product (aside from the removal of the signalpeptide). “Mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor protein” or“prepropeptide” or “preproprotein” all refer to the primary product oftranslation of mRNA; i.e., with pre- and propeptides still present. Pre-and propeptides may include, but are not limited to, intracellularlocalization signals. “Pre” in this nomenclature generally refers to thesignal peptide. The form of the translation product with only the signalpeptide removed but no further processing yet is called a “propeptide”or “proprotein.” The fragments or segments to be removed may themselvesalso be referred to as “propeptides.” A proprotein or propeptide thushas had the signal peptide removed, but contains propeptides (herereferring to propeptide segments) and the portions that will make up themature protein. The skilled artisan is able to determine, depending onthe species in which the proteins are being expressed and the desiredintracellular location, if higher expression levels might be obtained byusing a gene construct encoding just the mature form of the protein, themature form with a signal peptide, or the proprotein (i.e., a formincluding propeptides) with a signal peptide. For optimal expression inplants or fungi, the pre- and propeptide sequences may be needed. Thepropeptide segments may play a role in aiding correct peptide folding.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by assays that measure antipathogenic activity such asantifungal plate assays. See, for example, Duvick et al. (1992) J. Biol.Chem. 267:18841-18820, herein incorporated by reference.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more differentantipathogenic protein coding sequences can be manipulated to create anew antipathogenic protein possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled betweenthe antipathogenic protein gene of the invention and other knownantipathogenic protein genes to obtain a new gene coding for a proteinwith an improved property of interest, such as increased antifungalactivity. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The polynucleotides of the invention can be used to isolatecorresponding sequences from other organisms, particularly othermicroorganism, more particularly other fungi. In this manner, methodssuch as PCR, hybridization, and the like can be used to identify suchsequences based on their sequence homology to the sequences set forthherein. Sequences isolated based on their sequence identity to theentire sequences set forth herein or to variants and fragments thereofare encompassed by the present invention. Such sequences includesequences that are orthologs of the disclosed sequences. “Orthologs” isintended to mean genes derived from a common ancestral gene and whichare found in different species as a result of speciation. Genes found indifferent species are considered orthologs when their nucleotidesequences and/or their encoded protein sequences share at least 60%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orgreater sequence identity. Functions of orthologs are often highlyconserved among species. Thus, isolated polynucleotides that encode foran antipathogenic, particularly antifungal, protein and which hybridizeunder stringent conditions to the sequences disclosed herein, or tovariants or fragments thereof, are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the polynucleotides of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

For example, an entire polynucleotide disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding polynucleotides and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among antipathogenicpolynucleotide sequences and are optimally at least about 10 nucleotidesin length, and most optimally at least about 20 nucleotides in length.Such probes may be used to amplify corresponding polynucleotides from achosen organism by PCR. This technique may be used to isolate additionalcoding sequences from a desired organism or as a diagnostic assay todetermine the presence of coding sequences in an organism. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is intended to mean conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the thermal melting point (T_(m))can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seewww.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalentprogram” is intended to mean any sequence comparison program that, forany two sequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides, can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, and the like.

In some embodiments, expression cassettes comprising a promoter operablylinked to a heterologous nucleotide sequence of the invention thatencodes an antipathogenic polypeptide are further provided. Theexpression cassettes of the invention find use in generating transformedplants, plant cells, and microorganisms and in practicing the methodsfor inducing pathogen resistance disclosed herein. The expressioncassette will include 5′ and 3′ regulatory sequences operably linked toa polynucleotide of the invention. “Operably linked” is intended to meana functional linkage between two or more elements. For example, anoperable linkage between a polynucleotide of interest and a regulatorysequence (i.e., a promoter) is functional link that allows forexpression of the polynucleotide of interest. Operably linked elementsmay be contiguous or non-contiguous. When used to refer to the joiningof two protein coding regions, by operably linked is intended that thecoding regions are in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes. Such an expression cassette isprovided with a plurality of restriction sites and/or recombinationsites for insertion of the polynucleotide that encodes an antipathogenicpolypeptide to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter),translational initiation region, a polynucleotide of the invention, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theinvention may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theinvention may be heterologous to the host cell or to each other. As usedherein, “heterologous” in reference to a sequence is a sequence thatoriginates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The optionally included termination region may be native with thetranscriptional initiation region, may be native with the operablylinked polynucleotide of interest, may be native with the plant host, ormay be derived from another source (i.e., foreign or heterologous) tothe promoter, the polynucleotide of interest, the host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidsRes. 15:9627-9639. In particular embodiments, the potato proteaseinhibitor II gene (PinII) terminator is used. See, for example, Keil etal. (1986) Nucl. Acids Res. 14:5641-5650; and An et al. (1989) PlantCell 1:115-122, herein incorporated by reference in their entirety.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed organism. For example, the polynucleotidescan be synthesized using plant-preferred codons for improved expression.See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picomavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph. D. Thesis, University of Heidelberg; Reines et al. (1993) Proc.Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph. D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

A number of promoters can be used in the practice of the invention,including the native promoter of the polynucleotide sequence ofinterest. The promoters can be selected based on the desired outcome. Awide range of plant promoters are discussed in the recent review ofPotenza et al. (2004) In Vitro Cell Dev Biol—Plant 40:1-22, hereinincorporated by reference. For example, the nucleic acids can becombined with constitutive, tissue-preferred, pathogen-inducible, orother promoters for expression in plants. Such constitutive promotersinclude, for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

Generally, it will be beneficial to express the gene from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819, herein incorporated byreference.

Of interest are promoters that result in expression of a protein locallyat or near the site of pathogen infection. See, for example, Marineau etal. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) MolecularPlant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl.Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet.2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. Seealso, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc.Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J.3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No.5,750,386 (nematode-inducible); and the references cited therein. Ofparticular interest is the inducible promoter for the maize PRms gene,whose expression is induced by the pathogen Fusarium moniliforme (see,for example, Cordero et al. (1992) Physiol. Mol. Plant Path.41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expressionof the antipathogenic polypeptides of the invention within a particularplant tissue. For example, a tissue-preferred promoter may be used toexpress an antifungal polypeptide in a plant tissue where diseaseresistance is particularly important, such as, for example, the roots orthe leaves. Tissue-preferred promoters include Yamamoto et al. (1997)Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) PlantPhysiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol. Biol. 23(6): 1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Vascular tissue-preferred promoters are known in the art and includethose promoters that selectively drive protein expression in, forexample, xylem and phloem tissue. Vascular tissue-preferred promotersinclude, but are not limited to, the Prenus serotina prunasin hydrolasegene promoter (see, e.g., International Publication No. WO 03/006651),and also those found in U.S. Pat. No. 6,921,815.

Stalk-preferred promoters may be used to drive expression of anantipathogenic polypeptide of the invention. Exemplary stalk-preferredpromoters include the maize MS8-15 gene promoter (see, for example, U.S.Pat. No. 5,986,174 and International Publication No. WO 98/00533), andthose found in Graham et al. (1997) Plant Mol Biol 33(4): 729-735.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is a preferred endosperm-specificpromoter. Glob-1 is a preferred embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

In certain embodiments the nucleic add sequences of the presentinvention can be stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredphenotype. For example, the polynudeotides of the present invention maybe stacked with any other polynucleotides of the present invention, suchas any combination of SEQ ID NOS: 1, 3, 5,7, and 9, or with otherantifungal genes and the like. The combinations generated can alsoinclude multiple copies of any one of the polynucleotides of interest.The polynucleotides of the present invention can also be stacked withany other gene or combination of genes to produce plants with a varietyof desired trait combinations including but not limited to traitsdesirable for animal feed such as high oil genes (e.g., U.S. Pat. No.6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat. Nos.5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine(Williamson at al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122);and high inethionine proteins (Pedersen et al. (1986) J. Biol. Chem.261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989)Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modifiedstorage proteins (U.S. Pat. No. 6.858.778); and thioredoxins (U.S. Pat.No. 7,009,087, the disclosures of which are herein incorporated byreference. The polynucleotides of the present invention can also bestacked with traits desirable for insect, disease or herbicideresistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos.5,366,892; 5,747,450; 5,737,514; 5723,756; 5,593,881; Geiser et al(1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol.24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);avirulence and disease resistance genes (Jones et al. (1994) Science266:789; Martin at al. (1993) Science 262:1432; Mindrinos et al. (1994)Cell 78:1089); acetolactate synthase (ALS) mutants that lead toherbicide resistance such as the S4 and/or Hra mutations; inhibitors ofglutainine synthase such as phosphinothricin or basta (e.g., bar gene);and glyphosate resistance (EPSPS genes. GAT genes such as thosedisclosed in U.S. Patent Application Publication US2004/0082770, alsoWO02/36782 and WO03/092360)); and traits desirable for processing orprocess products such as high oil (e.g., U.S. Pat. No. 6,232,529);modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-COA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyallcanoates (PHAs)) the disclosures of which are hereinincorporated by reference. One could also combine the polynueleotides ofthe present invention with polynucleotides providing agronomic traitssuch as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalkstrength, flowering time, or transformation technology traits such ascell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364;WO 99/25821), the disclosures of which are herein incorporated byreference.

These stacked combinations can be created by any method including butnot limited to cross breeding plants by any conventional or TopCross®methodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide. In some embodiments, thepolynucleotide will be presented in such a manner that the sequencegains access to the interior of a cell of the plant, including itspotential insertion into the genome of a plant. The methods of theinvention do not depend on a particular method for introducing asequence into a plant, only that the polynucleotide gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotides into plants are known in the art including, but notlimited to, stable transformation methods, transient transformationmethods, and virus-mediated methods. Polypeptides can also be introducedto a plant in such a manner that they gain access to the interior of theplant cell or remain external to the cell but in close contact with it.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” or “transient expression” is intended to meanthat a polynucleotide is introduced into the plant and does notintegrate into the genome of the plant or a polypeptide is introducedinto a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055- and 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, Sanford et al., U.S. Pat. Nos.4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborgand Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Alsosee Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al.(1987) Particulate Science and Technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the antipathogenic sequences of the inventioncan be provided to a plant using a variety of transient transformationmethods. Such transient transformation methods include, but are notlimited to, the introduction of the antipathogenic protein or variantsand fragments thereof directly into the plant or the introduction of theantipathogenic protein transcript into the plant. Such methods include,for example, microinjection or particle bombardment. See, for example,Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986)Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush et al. (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the polynucleotide can be transiently transformed intothe plant using techniques known in the art. Such techniques includeviral vector system and the precipitation of the polynucleotide in amanner that precludes subsequent release of the DNA. Thus, thetranscription from the particle-bound DNA can occur, but the frequencywith which it's released to become integrated into the genome is greatlyreduced. Such methods include the use particles coated withpolyethylimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. It is recognized that the an antipathogenic polypeptide of theinvention may be initially synthesized as part of a viral polyprotein,which later may be processed by proteolysis in vivo or in vitro toproduce the desired recombinant protein. Further, it is recognized thatpromoters of the invention also encompass promoters utilized fortranscription by viral RNA polymerases. Methods for introducingpolynucleotides into plants and expressing a protein encoded therein,involving viral DNA or RNA molecules, are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367,5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221;herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant have stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Pedigree breeding starts with the crossing of two genotypes, such as anelite line of interest and one other elite inbred line having one ormore desirable characteristics (i.e., having stably incorporated apolynucleotide of the invention, having a modulated activity and/orlevel of the polypeptide of the invention, etc) which complements theelite line of interest. If the two original parents do not provide allthe desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneous lines asa result of self-pollination and selection. Typically in the pedigreemethod of breeding, five or more successive filial generations ofselfing and selection is practiced: F1→F2; F2→F3; F3→F4; F4→F₅, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed inbred. In specificembodiments, the inbred line comprises homozygous alleles at about 95%or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify anelite line of interest and a hybrid that is made using the modifiedelite line. As discussed previously, backcrossing can be used totransfer one or more specifically desirable traits from one line, thedonor parent, to an inbred called the recurrent parent, which hasoverall good agronomic characteristics yet lacks that desirable trait ortraits. However, the same procedure can be used to move the progenytoward the genotype of the recurrent parent but at the same time retainmany components of the non-recurrent parent by stopping the backcrossingat an early stage and proceeding with selfing and selection. Forexample, an F1, such as a commercial hybrid, is created. This commercialhybrid may be backcrossed to one of its parent lines to create a BC1 orBC2. Progeny are selfed and selected so that the newly developed inbredhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the non-recurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newhybrids and breeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of maize inbred line of interest, comprising thesteps of crossing a plant of maize inbred line of interest with a donorplant comprising a mutant gene or transgene conferring a desired trait(i.e., increased pathogen resistance), selecting an F1 progeny plantcomprising the mutant gene or transgene conferring the desired trait,and backcrossing the selected F1 progeny plant to the plant of maizeinbred line of interest. This method may further comprise the step ofobtaining a molecular marker profile of maize inbred line of interestand using the molecular marker profile to select for a progeny plantwith the desired trait and the molecular marker profile of the inbredline of interest. In the same manner, this method may be used to producean F1 hybrid seed by adding a final step of crossing the desired traitconversion of maize inbred line of interest with a different maize plantto make F1 hybrid maize seed comprising a mutant gene or transgeneconferring the desired trait.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and topcrossing. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation breeding is one of many methods that could be used to introducenew traits into an elite line. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development” Fehr, 1993Macmillan Publishing Company the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of elite lines that comprisessuch mutations.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plant can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

The present invention may be used to induce pathogen resistance orprotect from pathogen attack any plant species, including, but notlimited to, monocots and dicots. Examples of plant species of interestinclude, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B.napus, B. rapa, B. juncea), particularly those Brassica species usefulas sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa),rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet(e.g., pearl millet (Pennisetum glaucum), proso millet (Panicummiliaceum), foxtail millet (Setaria italica), finger millet (Eleusinecoracana)), sunflower (Helianthus annuus), safflower (Carthamustinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweetpotato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Coffeaspp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrustrees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis),banana (Musa spp.), avocado (Persea americana), fig (Ficus casica),guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Antipathogenic compositions, particularly antifungal compositions, arealso encompassed by the present invention. Antipathogenic compositionsmay comprise antipathogenic polypeptides or transformed microorganismscomprising a nucleotide sequence that encodes an antipathogenicpolypeptide. The antipathogenic compositions of the invention may beapplied to the environment of a plant pathogen, as described hereinbelow, thereby protecting a plant from pathogen attack. Moreover, anantipathogenic composition can be formulated with an acceptable carrierthat is, for example, a suspension, a solution, an emulsion, a dustingpowder, a dispersible granule, a wettable powder, and an emulsifiableconcentrate, an aerosol, an impregnated granule, an adjuvant, a coatablepaste, and also encapsulations in, for example, polymer substances.

A gene encoding an antipathogenic, particularly antifungal, polypeptideof the invention may be introduced into any suitable microbial hostaccording to standard methods in the art. For example, microorganismhosts that are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops ofinterest may be selected. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, and to provide for stable maintenance andexpression of the gene expressing the antifungal protein.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli andAzotobacter vinlandir and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

Other illustrative prokaryotes, both Gram-negative and gram-positive,include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium;Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas,Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae,such as Pseudomonas and Acetobacter; Azotobacteraceae andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Microbial host organisms of particular interest include yeast, such asRhodotorula spp., Aureobasidium spp., Saccharomyces spp., andSporobolomyces spp., phylloplane organisms such as Pseudomonas spp.,Erwinia spp., and Flavobacterium spp., and other such organisms,including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis,and the like.

Genes encoding the antifungal proteins of the invention can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver antifungal proteins to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the antifungal polypeptides of the inventioncan be introduced into a root-colonizing Bacillus cereus by standardmethods known in the art.

Genes encoding antifungal proteins can be introduced, for example, intothe root-colonizing Bacillus by means of electrotransformation.Specifically, genes encoding the antifungal proteins can be cloned intoa shuttle vector, for example, pHT3101 (Lerecius et al. (1989) FEMSMicrobiol. Letts. 60: 211-218. The shuttle vector pHT3101 containing thecoding sequence for the particular antifungal protein gene can, forexample, be transformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218).

Methods are provided for protecting a plant from a pathogen comprisingapplying an effective amount of an antipathogenic protein or compositionof the invention to the environment of the pathogen. “Effective amount”is intended to mean an amount of a protein or composition sufficient tocontrol a pathogen. The antipathogenic proteins and compositions can beapplied to the environment of the pathogen by methods known to those ofordinary skill in the art.

The antifungal compositions of the invention may be obtained by theaddition of a surface-active agent, an inert carrier, a preservative, ahumectant, a feeding stimulant, an attractant, an encapsulating agent, abinder, an emulsifier, a dye, a UV protective, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetpathogens. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the present invention are normally applied in theform of compositions and can be applied to the crop area, plant, or seedto be treated. For example, the compositions of the present inventionmay be applied to grain in preparation for or during storage in a grainbin or silo, etc. The compositions of the present invention may beapplied simultaneously or in succession with other compounds. Methods ofapplying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the antipathogenic proteins, more particularly antifungalproteins, of the present invention include, but are not limited to,foliar application, seed coating, and soil application. The number ofapplications and the rate of application depend on the intensity ofinfestation by the corresponding pest or pathogen.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The antipathogenic compositions of the present invention can be in asuitable form for direct application or as a concentrate of primarycomposition that requires dilution with a suitable quantity of water orother diluant before application. The concentration of theantipathogenic polypeptide will vary depending upon the nature of theparticular formulation, specifically, whether it is a concentrate or tobe used directly. The composition contains 1 to 98% of a solid or liquidinert carrier, and 0 to 50%, optimally 0.1 to 50% of a surfactant. Thesecompositions will be administered at the labeled rate for the commercialproduct, optimally about 0.01 lb-5.0 lb. per acre when in dry form andat about 0.01 pts.-10 pts. per acre when in liquid form.

In a further embodiment, the compositions, as well as the transformedmicroorganisms and antipathogenic proteins, of the invention can betreated prior to formulation to prolong the antipathogenic, particularlyantifungal, activity when applied to the environment of a targetpathogen as long as the pretreatment is not deleterious to the activity.Such treatment can be by chemical and/or physical means as long as thetreatment does not deleteriously affect the properties of thecomposition(s). Examples of chemical reagents include but are notlimited to halogenating agents; aldehydes such a formaldehyde andglutaraldehyde; anti-infectives, such as zephiran chloride; alcohols,such as isopropanol and ethanol; and histological fixatives, such asBouin's fixative and Helly's fixative (see, for example, Humason (1967)Animal Tissue Techniques (W. H. Freeman and Co.).

The antipathogenic compositions of the invention can be applied to theenvironment of a plant pathogen by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pathogen has begun to appearor before the appearance of pathogens as a protective measure. Forexample, the antipathogenic protein and/or transformed microorganisms ofthe invention may be mixed with grain to protect the grain duringstorage. It is generally important to obtain good control of pathogensin the early stages of plant growth, as this is the time when the plantcan be most severely damaged. The compositions of the invention canconveniently contain an insecticide if this is thought necessary. In oneembodiment of the invention, the composition is applied directly to thesoil, at a time of planting, in granular form of a composition of acarrier and dead cells of a Bacillus strain or transformed microorganismof the invention. Another embodiment is a granular form of a compositioncomprising an agrochemical such as, for example, a herbicide, aninsecticide, a fertilizer, an inert carrier, and dead cells of aBacillus strain or transformed microorganism of the invention.

Compositions of the invention find use in protecting plants, seeds, andplant products in a variety of ways. For example, the compositions canbe used in a method that involves placing an effective amount of theantipathogenic, more particularly, antifungal, composition in theenvironment of the pathogen by a procedure selected from the groupconsisting of spraying, dusting, broadcasting, or seed coating.

Before plant propagation material (fruit, tuber, bulb, corm, grains,seed), but especially seed, is sold as a commercial product, it iscustomarily treated with a protective coating comprising herbicides,insecticides, fungicides, bactericides, nematicides, molluscicides, ormixtures of several of these preparations, if desired together withfurther carriers, surfactants, or application-promoting adjuvantscustomarily employed in the art of formulation to provide protectionagainst damage caused by bacterial, fungal, or animal pests. In order totreat the seed, the protective coating may be applied to the seedseither by impregnating the tubers or grains with a liquid formulation orby coating them with a combined wet or dry formulation. In addition, inspecial cases, other methods of application to plants are possible,e.g., treatment directed at the buds or the fruit.

The plant seed of the invention comprising a DNA molecule comprising anucleotide sequence encoding an antipathogenic polypeptide of theinvention may be treated with a seed protective coating comprising aseed treatment compound, such as, for example, captan, carboxin, thiram,methalaxyl, pirimiphos-methyl, and others that are commonly used in seedtreatment. Alternatively, a seed of the invention comprises a seedprotective coating comprising an antipathogenic, more particularlyantifungal, composition of the invention is used alone or in combinationwith one of the seed protective coatings customarily used in seedtreatment.

The antifungal polypeptides of the invention can be used for anyapplication including coating surfaces to target microbes. In thismanner, the target microbes include human pathogens or microorganisms.Surfaces that might be coated with the antifungal polypeptides of theinvention include carpets and sterile medical facilities. Polymer boundpolypeptides of the invention may be used to coat surfaces. Methods forincorporating compositions with antimicrobial properties into polymersare known in the art. See U.S. Pat. No. 5,847,047, herein incorporatedby reference.

The embodiments of the present invention may be effective against avariety of plant pathogens, particularly fungal pathogens, such as, forexample, Colletotrichum graminicola, Diplodia maydis, Fusariumgraminearum, and Fusarium verticillioides. Pathogens of the inventioninclude, but are not limited to, viruses or viroids, bacteria, insects,nematodes, fungi, and the like. Viruses include any plant virus, forexample, tobacco or cucumber mosaic virus, ringspot virus, necrosisvirus, maize dwarf mosaic virus, etc. Fungal pathogens, include but arenot limited to, Colletotrichum graminicola, Diplodia maydis, Fusariumgraminearum, and Fusarium verticillioides. Specific pathogens for themajor crops include: Soybeans: Phytophthora megasperma fsp. glycinea,Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum,Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae),Diaporthe phaseolorum var. caulivora, Sclerotium roftsii, Cercosporakikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichumdematium (Colletotichum truncatum), Corynespora cassiicola, Septoriaglycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonassyringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli,Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybeanmosaic virus, Glomerella glycines, Tobacco Ring spot virus, TobaccoStreak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythiumultimum, Pythium debaryanum, Tomato spotted wilt virus, Heteroderaglycines Fusarium solani; Canola: Albugo candida, Alternaria brassicae,Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum,Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica,Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganesesubsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythiumsplendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthoramegasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis,Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochilamedicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonascampestris p.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum,Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulinabriosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonosporameliloti, Stemphylium botryosum, Leptotrichila medicaginis; Wheat:Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonascampestris p.v. translucens, Pseudomonas syringae p.v. syringae,Alternaria alternata, Cladosporium herbarum, Fusarium graminearum,Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochytatritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphegraminis f.sp. tritici, Puccinia graminis f.sp. tritici, Pucciniarecondita f.sp. tritici, Puccinia striiformis, Pyrenophoratritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae,Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctoniacerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, BarleyYellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus,Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American WheatStriate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis,Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythiumarrhenomannes, Pythium gramicola, Pythium aphanidermatum, High PlainsVirus, European wheat striate virus; Sunflower: Plasmopora halstedii,Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsishelianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea,Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum,Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Pucciniahelianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora,Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis;Corn: Colletotrichum graminicola, Fusarium moniliforme var.subglutinans, Erwinia stewartii, F. verticillioides, Gibberella zeae(Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythiumirregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens,Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolarismaydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I,II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganense subsp.nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora,Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, C.sublineolum, Cercospora sorghi, Gloeocercospora sorghi, Ascochytasorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestrisp.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea,Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme,Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola,Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonasalboprecipitans), Ramulispora sorghi, Ramulispora sorghicola,Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana),Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, MaizeDwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani,Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi,Peronosclerospora philippinensis, Sclerospora graminicola, Fusariumgraminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythiumgraminicola, etc.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

The following examples are provided by way of illustration, not by wayof limitation.

EXPERIMENTAL

Methods of growing fungal cultures are well known in the art. Forsubculturing the fungal cultures disclosed herein, any broth generallysuitable for growing fungi may be used, including, for example, potatodextrose broth infra (Becton Dickinson Microbiology Systems, Sparks,Md.), Czapek-Dox broth infra (Becton Dickinson Microbiology Systems,Sparks Md.), Sabouraud broth (BBL #210986, Voigt Global DistributionLLC, Kansas City, Mo.), and the like.

Example 1 Isolation of Antifungal Polypeptide LBNL-5220 (SEQ ID NO:1)

An environmental sample was collected from a geothermal mud pool (GPSdata: 54°26′256″×160°08″385″) located in the Kronotsky National Park inKamchatka, Russia. The organism, denoted herein as K01-17A5, thatproduced the antifungal polypeptide LBNL-5220, was isolated from diversecolonies that grew on potato dextrose agar using isolation techniquesand media as published earlier (Hunter-Cevera, J. C. and Belt, A. 1999.Isolation of Cultures, Manual of Industrial Microbiology andBiotechnology, Chapter 1, pp. 3-20). The molecular-levelcharacterization, based on whole-cell fatty acid methyl ester (FAME)analysis and on sequencing of the D1/D2 domains of the large subunitribosomal RNA-coding genes, was confirmed by classical phenotype-basedidentification. The strain was identified as the ascomycetous fungusPenicillium simplicissimum (Oudemans) Thom (syn. P. janthinellumBiourge).

A designed set of specific growth conditions, i.e., nutrient content,temperature, pH, incubation time, aeration, etc., were applied to theisolated fungus to promote the production of secondary metabolites andnovel natural products. Biomass and supernatant of the resultingmicrobial fermentation were then separated by centrifugation. Thecell-free supernatant LBNL-5220 was assayed to determine the presence ofheat labile antifungal activity. After confirming that heat labileantifungal activity was present in the LBNL-5220 supernatant, thecell-free supernatant of a large scale, 500-ml culture was provided andsubjected to solid phase extraction, as described below.

Oasis HLB extraction cartridges (6 gram, 35 mL) (Waters Corporation,Milford, Mass.) were used for solid phase extraction (SPE).Specifically, the SPE cartridge was made wet with one cartridge volumeof methanol and then conditioned with approximately 40 mL Solvent A (2%acetonitrile, 0.1% TFA). The extract was treated with 5× solvent A to afinal concentration of 1× and centrifuged for 20 min at 3,000×g. Thesupernatant was loaded onto an SPE cartridge, and the SPE cartridge waswashed with approximately 40 mL solvent A. The SPE cartridge was elutedwith approximately 40 mL 90% acetonitrile, 0.1% TFA. The eluted samplewas partially dried in a centrifugal evaporator (Speed Vac) and frozenwith liquid nitrogen and lyophilized to dryness.

The dried extract was re-suspended in deionized H₂O (0.5 mL: 12.5 mLstarting culture filtrate), and the re-suspended extract was enrichedfor proteins using a Sephadex G10 (Amersham Biosciences AB, Uppsala,Sweden) spin column. Bio-Spin disposable chromatography columns (Bio-RadLaboratories, Hercules Calif.) were filled to approximately 0.75 mL bedvolume with Sephadex G10 that had been pre-equilibrated in phosphatebuffered saline (PBS) and were centrifuged for 1 minute at 1,000×g.10×PBS was added to the SPE extract to a final concentration of 1×PBS.200 μL of SPE extract in PBS was added to each pre-spun Bio-Spin column,and loaded Bio-Spin columns were centrtifuged for 5 minutes at 1,000×gto elute proteins.

Sephadex G-10 treated extracts were tested for antifungal activityagainst FVE, CGR, FGR, and DMA using an antifungal plate assay, asdescribed in Example 3. Assays were performed in ½ area 96 well clearbottom plates. FVE, FGR and DMA were tested at 4,000 spores/mL in ¼×potato dextrose broth (Becton Dickinson Microbiology Systems, Sparks,Md.). CGR was tested at 4,000 spores/mL in ¼× Czapek-Dox (BectonDickinson Microbiology Systems, Sparks Md.)+180 mL/L V8 juice. Cultureswere allowed to develop at 27° C. for 24 hours. Assays were scored byvisualizing fungal growth with an inverted microscope.

Antifungal extracts were fractionated by HPLC with a Jupiter 5μ C5 300 Å150×4.6 mm column (Phenomenex, Torrance, Calif.). HPLC startingconditions were 5% acetonitrile, 0.05% heptafluorobutyric acid (HFBA),0.4 mL/minute. Following injection, the flow rate was raised to 0.8mL/minute over 1 minute. After an additional minute, a 94 minuteexponentially curved gradient (Waters gradient curve 7, WatersCorporation, Milford, Mass.) was started to 86% acetonitrile, 0.05%HFBA. 30 second fractions were collected into ½ area 96 well clearbottom assay plates. Plates containing fractionated extracts were thendried in a centrifugal evaporator. The dried fractionated extracts werethen screened for antifingal activity as described above. The HPLCfractions from approximately 64 to 66 minutes were found to haveantifungal activity against FVE, CGR, FGR and DMA.

Additional HPLC fractionations were performed to bulk up the antifungalfraction. This bulked up antifungal fraction was further purified usingμ-bore HPLC with a Zorbax 3.5μ C8 300 Å 150×1.0 mm column (AgilentTechnologies, Palo Alto, Calif.). Starting conditions were 5%acetonitrile, 0.1% trifluoroacetic acid (TFA), 50 μL/minute. Followingsample injection a 40 minute linear gradient was started to 23%acetonitrile, 0.1% TFA. Fractions were collected manually, dried in acentrifugal evaporator and assayed for antifungal activity as describedabove. A peak eluting at approximately 27 minutes was found to havebroad spectrum activity against FVE, CGR, FGR and DMA. ESI mass spectrawere obtained on a Finnigan LCQ mass spectrometer by directly infusingthe purified sample at 1 μL/minute.

Reduction and alkylation was required for efficient N-terminalsequencing. Approximately 10 μg dried protein was re-suspended into 18μL 0.1 M ammonium bicarbonate, 8 M urea pH 8.3. This solution wastransferred to limited volume HPLC autosampler vial. 1 μL 200 mM DTT wasadded and the solution was incubated at 50° C. for 1 hour. Subsequently,1 μL 500 mM iodoacetamide was added, and the solution was incubated at37° C. for 30 minutes in the dark. The iodoacetamide alkylation was thenquenched by adding 2 μL 25% trifluoroacetic acid. The alkylated proteinwas purified by μ-bore HPLC on a Vydac C4 150×1.0 mm column. A 100minute linear gradient was performed from 5% acetonitrile, 0.1% TFA to95% acetonitrile 0.1% TFA. The column flow rate was 50 μL/minute.

N-terminal Sequencing

The full elucidation of the sequence set forth in SEQ ID NO:1 byN-terminal sequencing required sequencing of Asp-N (Calbiochem) digestedLBNL-5220 fragments. 5 μL (0.2 μg) Asp-N was added to approximately 10μg LBNL-5220 that was dissolved in 15 μL 50 mM sodium phosphate 0.5 Murea pH 7.5. This solution was incubated for approximately 14 hours at37° C. The digested fragments were purified by μ-bore HPLC with a Zorbax3.5μ C8 300 Å 150×1.0 mm column (Agilent Technologies, Palo Alto,Calif.). Starting conditions were 5% acetonitrile, 0.1% trifluoroaceticacid (TFA), 50 μL/minute. Following sample injection a 100 minute lineargradient was started to 95% acetonitrile, 0.1% TFA.

Example 2 Isolation of Antifungal Polypeptides LBNL-5197/8-1 andLBNL-5197/8-2 (SEQ ID NOs:3 and 5) and LBNL-09827 (SEQ ID NOs:7 and 9)

The two distinct strains of another organism, denoted herein as K01-17A2and K01-17A4, that produced the antifungal polypeptides LBNL-5197/8-1and LBNL-5197/8-2 (SEQ ID NOs:3 and 5), respectively, were isolated froman environmental sample that was collected as in Example 1. Theorganisms were selected from diverse colonies that grew on potatodextrose agar. The molecular-level characterization of the strains,based on whole-cell fatty acid methyl ester (FAME) analysis and onsequencing of the D1/D2 domains of the large subunit ribosomalRNA-coding genes, was confirmed by classical phenotype-basedidentification. The two organisms were identified as strains of theascomycetous fungus Penicillium miczyinskii Zaleski (syn. P. soppiZaleski).

A designed set of specific growth conditions, i.e., nutrient content,temperature, pH, incubation time, aeration, etc., were applied to theisolated fungi to promote the production of secondary metabolites andnovel natural products. Biomass and supernatants of the resultingmicrobial fermentations were then separated by centrifugation. Thecell-free supernatants LBNL-5197 (K01-17A2) and LBNL-5198 (K01-17A4)were assayed to determine the presence of heat labile antifungalactivity. After confirming that heat labile antifungal activity waspresent in the LBNL-5197 (K01-17A2) and LBNL-5198 (K01-17A4)supernatants, the cell-free supernatants of a large scale, 500-mlcultures were provided and subjected to solid phase extraction, asdescribed in Example 1.

Another fungal organism of interest, IMV 00127, that produced theantifungal polypeptides LBNL-9827-1 and LBNL-9827-2 (SEQ ID NOs:7 and9), was isolated on potato dextrose agar (PDA) from an apparentlycontaminated fodder grain sample that was collected in the Kharkovregion, Ukraine. The pure culture of the organism was previouslymaintained at the D. K. Zabolotny Institute of Microbiology andVirology, Kiev, Ukraine, at room temperature on malt extract agar slantby sub-culturing it in regular intervals. The strain IMV 00127 wasidentified as Monascus ruber by employing classical fungal taxonomicmethods and molecular-level techniques, such as fatty acid methyl ester(FAME) analysis and sequencing of the D1/D2 domain of the large subunitrRNA-coding gene.

A designed set of specific growth conditions, i.e., nutrient content,temperature, pH, incubation time, aeration, etc., were applied to theisolated fungus to promote the production of secondary metabolites andnovel natural products. Biomass and supernatant of the resultingmicrobial fermentation was then separated by centrifugation. Thecell-free supernatant LBNL-9827 was assayed to determine the presence ofheat labile antifungal activity. After confirming that heat labileantifungal activity was present in the LBNL-9827 supernatant, thecell-free supernatant of a large scale, 500-ml culture was provided andsubjected to solid phase extraction, as described in Example 1.

Antifungal extracts were fractionated by HPLC as described in Example 1,and antifungal activity against FVE, CGR, FGR and DMA was found in the62-68 minute HPLC fractions.

Additional HPLC fractionations were performed to bulk up the antifungalfraction as described in Example 1. Two peaks containing anti-FVEactivity were identified which eluted at approximately 23 (i.e., SEQ IDNO:3) and 31 minutes (i.e., SEQ ID NO:5), respectively. ESI mass spectrawere obtained on a Finnigan LCQ mass spectrometer by directly infusingthe purified sample at 1 μL/minute.

Reduction and alkylation was performed to prepare samples for efficientN-terminal sequencing as described in Example 1. The alkylated proteinwas purified by μ-bore HPLC on a Zorbax 3.5μ C8 300 Å 150×1.0 mm column(Agilent Technologies, Palo Alto, Calif.). Starting conditions were 5%acetonitrile, 0.1% trifluoroacetic acid (TFA), 50 μL/minute. 10 minutesafter sample injection a 50 minute linear gradient was started to 95%acetonitrile, 0.1% TFA.

N-terminal Sequencing of SEQ ID NO:3

Further elucidation of the Peak #1 sequence (SEQ ID NO:3) by N-terminalsequencing required sequencing digested Peak #1 fragments. Asp-N wasused to prepare digested fragments for sequencing. 5 μL 0.2 μg Asp-N wasadded to approximately 10 μg Peak #1 that was dissolved in 15 μL 50 mMsodium phosphate 0.5 M urea pH 7.5. This solution was incubated forabout 14 hours at 37° C. The digested fragments were purified by μ-boreHPLC with a Zorbax 3.5% C8 300 Å 150×1.0 mm column (AgilentTechnologies, Palo Alto, Calif.). Starting conditions were 5%acetonitrile, 0.1% trifluoroacetic acid (TFA), 50 μL/minute. Followingsample injection a 100 minute linear gradient was started to 95%acetonitrile, 0.1% TFA.

Example 3 Antifungal Activity Assays

The antifungal activity of the polypeptides of the invention against thefungal pathogens Fusarium verticillioides (FVE), Colletotrichumgraminicola (CGR), Fusarium graminearum (FGR) and Diplodia maydis (DMA)was assessed using a standard plate assay. Silica gel stocks of eachfungus were stored at −20° C. prior to use.

Preparation of Cultures for Spore Production

Cultures of FVE were prepared using V8 agar plates. FGR, CRG, and DMAcultures were prepared using ½× oatmeal agar. Media recipes are providedbelow.

Specifically, tubes containing silica-gel fungal stocks stored at −20°C. were briefly flamed, and approximately 5 crystals were sprinkled ontothe agar surface. 2-3 plates of each fungal isolate were prepared. Thenewly plated cultures were stored in a plastic box in the dark at roomtemperature to prevent the cultures from drying out. New cultures wereprepared every other week to maintain a consistent supply of spores.

Spore Preparation

Spores were prepared from 2-4 week old cultures of FVE, FGR, CGR, andDMA. For FGR, FVE, and DMA, a portion of the culture plate was rinsedwith a small amount of assay medium. The rinse solution was permitted toremain on the DMA plates for a time sufficient to allow the pycnidiarupture. The assay medium was then transferred to a sterile tube.Samples were vortexed, and spores were quantitated using ahemacytometer.

For CGR, a sterile loop was gently dragged across orange areas of theculture plate. The loop was then inserted into a small volume of assaymedia, and the media was mixed with the loop to suspend the spores.Samples were vortexed, and spores were quantitated using ahemacytometer.

Spores were diluted to the desired concentration with assay medium(4,000 spores per mL for FGR, FVE, and CGR, and 6,000 spores per mL forDMA) and kept on ice prior to beginning the antifungal activity assay.

Assay Plate Preparation Details

Standard non-tissue culture treated 96 well flat bottom plates or ½ areanon-treated plates (Costar) were used in the antifungal plate assays.Assay medium was ¼× potato dextrose broth for FVE, FGR and DMA, and ¼×Czapec-Dox V8 was used for CGR.

Antifungal polypeptides at various concentrations were added to theplates at 50 μL/well for a standard assay plate or 25 μL/well for a halfarea plate. An equal volume of media with fungal spores at 2 times theabove concentrations was then added to start the assay. AlternativelyHPLC fractionated lead samples were assayed by adding media with fungalspores (as above) into assay plates that the HPLC samples had been driedinto (Savant Speed-vac). The plates were sealed with a gas permeablemembrane (“Breathe-Easy”, Cat. No. BEM-1, Diversified Biotech, Boston,Mass.), and the assay was allowed to develop in the dark at 28° C. for24 to 48 hours.

After the incubation period, the plates were placed on an invertedmicroscope, and each well was examined and scored on a scale of 0-4,according to the following parameters: 0=no inhibition of fungal growthwhen compared to the negative control, 0.5=slight inhibition (overallgrowth is less than the negative control but growth from individualspores is not distinct), 1=slight inhibition (overall growth is lessthan the negative control but growth from individual spores is apparent,albeit not quite confluent), 2=moderate inhibition (growth from 1 sporecan easily be identified and is significantly less abundant than thenegative control; growth from each spore tends to look spherical),3=strong inhibition (spores have germinated but growth is limited to afew branches of short hyphae), 4=complete inhibition (spores have notgerminated. See, for example, Duvick et al. (1992) J. Biol. Chem. 267:18814-18820). A score sheet containing representative examples of eachlevel of antifungal activity is provided in FIG. 3.

Results

FIG. 4 provides the photographic results of antifungal activity assayswith the polypeptide set forth in SEQ ID NO:1. This polypeptide showedantifungal activity against FVE, FGR, CGR, and DMA.

FIGS. 5A and 5B provide the antifungal activity profiles obtained inassays with the polypeptides set forth in SEQ ID NO:3 and SEQ ID NO:9,respectively. Both polypeptides inhibited FVE, FGR, CGR, and DMA in adose-dependent fashion.

HPLC purified LBNL-9827-1 polypeptide (SEQ ID NO:7) was also tested inantifungal assays against FVE. The results of these experimentsindicated that this polypeptide is active against FVE (data not shown).Notably, SEQ ID NO:7 is identical to SEQ ID NO:9 except that SEQ ID NO:9comprises 3 additional N-terminal amino acids.

Media Recipes

1× Czapek-Dox V8 Broth

For each liter, suspend 35 grams Difco Czapek-Dox Broth (#233810) indH₂O and add 180 milliliters V8 juice that has been clarified bycentrifugation (3,000×g is plenty). Raise final volume to 1 liter andautoclave at 121° C. for 20 minutes. The media is filter sterilized toremove any remaining debris.

1× Potato Dextrose Broth

For each liter, suspend 24 grams Difco Potato Dextrose Broth(#0549-17-9) in dH₂O and raise final volume to 1 liter and autoclave at121° C. for 20 minutes. The media is filter sterilized to remove anyremaining debris.

CCM (Cochliobolus Complete Medium)

Solution A: 10 grams Ca(NO₃)₂.4H₂O per 100 mL

Solution B: 2 grams KH₂PO₄+1.5 grams NaCl per 100 mL. Adjust pH to 5.3with NaOH

Solution C: 2.5 grams MgSO₄.7H₂O per 100 mL

Put 900 mL dH₂O into vessel on stir plate. Add to the water in order andallow each component to dissolve before proceeding to the next step:

-   -   10 mL solution A    -   10 mL solution B    -   10 mL solution C    -   10 grams glucose    -   1 gram Difco yeast extract    -   0.5 gram casein hydrolysate (acid)    -   0.5 gram casein hydrolysate (enzyme)

Bring final volume to 1 liter and filter sterilize, but do notautoclave. ¼×CCM-phosphate is made by diluting the 1×CCM medium to ¼×with 10 mM sodium phosphate buffer pH 5.8

V8 Agar

For each liter, dissolve 180 mL V8 juice and 3 grams calcium carbonatein 820 mL deionized water and then add 17 grams Bacto-agar in dH₂O in a4 liter vessel. 10 drops of 5% antifoam A may be optionally added perliter prepared. Cover and autoclave at 121° C. for 20 minutes. Pourplates in sterile hood.

Oatmeal Agar

For each liter, suspend 36.24 grams of Difco Oatmeal Agar (#0552-17-3)and 4.25 grams agar in dH₂O in a 4 liter vessel, cover and autoclave at121° C. for 20 minutes. Pour plates in sterile hood.

FVE FGR CGR DMA Isolate name MO33 73B ISU Carroll-IA-99 Warren-IN-96Medium for V8 Agar 1/2X Oatmeal Agar 1/2X Oatmeal Agar 1/2X Oatmeal Agarsporulation Agar Agar Agar Agar culture age 2-4 weeks old 2-4 weeks old2-4 weeks old 2-4 weeks old range for in vitro assay Suggested Everyother Every other Every other Every other schedule for week week weekweek starting agar cultures Liquid medium ¼ x potato ¼ x potato ¼ xCzapec- ¼ x potato for in vitro assay dextrose broth dextrose broth DoxV8 broth dextrose broth Spore Density 4,000 4,000 4,000 6,000 for invitro assay (spores/mL)

Example 4 Identification of SEQ ID NO: 11 and 15 from a ComputerHomology Search

Gene identities can be determined by conducting BLAST (Basic LocalAlignment Search Tool; Altschul, S. F., et al. (1993) J. Mol. Biol.215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches underdefault parameters for similarity to sequences contained in the BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases).

The polynucleotide sequences of the invention (i.e. SEQ ID NOs: 1, 3, 5,7 and 9) were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithm. TheDNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish, W. and States, D. J.Nature Genetics 3:266-272 (1993)) provided by the NCBI.

Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

SEQ ID NO: 11 was identified from a proprietary Pioneer database and wasconcluded to originate from an unspecified fungi endemic to maize (corn)plants. The sequence was found to be related to those sequences alreadyidentified from testing of the microbial fermentation broths and shownto have antifungal activity (i.e. SEQ ID NOs:1, 3, 5, 7 and 9). SEQ IDNO: 11 is clearly a member of the same family of antifungal proteins.

SEQ ID NO: 15 was identified from a public sequence database, however,the gene was not specifically identified in the available sequence; thatis, the ORF was not predicted nor annotated by the public domain. Wefound and identified the gene and its ORF encoding a peptide sequence,which was clearly related to the other known, tested sequences of theinvention. The identified sequence (SEQ ID NO: 15) clearly belongs tothe same family of antifungal proteins.

Example 5 Sequence Analysis of Antifungal Polypeptides

FIG. 1 shows an alignment of the amino acid sequences set forth in SEQID NOs:1, 3, 5, 7, and 9 with various known polypeptides sharingsequence similarity with the antipathogenic polypeptides of theinvention. Moreover, tables summarizing the global identity andsimilarity data are presented in Table 1A and Table 1B. Percent identityand similarity values were calculated using GAP with all defaultparameters, except that penalizing end gaps parameter was selected. SeeFIG. 2 for alignment limited to the novel sequences disclosed herein.

TABLE 1A Global Identity Table A. A. LBNL- LBNL- P. Unk_cds gignateusniger _(—) 5197-8-1 LBNL- LBNL- 9827-2 chrysogenum 2f.pk001.18 AFPunknown Fg_contig (SEQ 5197-8-2 9827-1 (SEQ AFP (SEQ (SEQ ID SEQ ID 196(SEQ ID (SEQ ID (SEQ ID ID (SEQ ID ID NO:59) NO:61) ID NO:17) NO:3)NO:5) NO:7) NO:9) NO:60) NO:13) LBNL-05220 49.02 35.19 52.73 48.15 40.7435.18 33.33 58.19 35.19 (SEQ ID NO:1) A. giganteus — 31.37 45.10 52.0035.29 35.29 35.29 48.08 31.37 AFP (SEQ ID NO:59) A. niger _(—) — — 33.3338 60 81.03 79.31 79.31 36.36 87.93 unknown (SEQ ID NO:61) Fg_contig196— — — 44.44 35.19 35.19 33.33 61.82 35.19 (SEQ ID NO:17) LBNL-5197- — —— — 36.84 38.60 40.35 57.41 42.11 8-1 (SEQ ID NO:3) LRNL-5197- — — — — —84.48 84.48 38.18 82.76 8-2 (SEQ ID NO:5) LBNL-9827-1 — — — — — —100.00  36.36 81.03 (SEQ ID NO:7) LBNL-9827-2 — — — — — — — 36.36 81.03(SEQ ID NO:9) P. — — — — — — — — 38.18 chrysogenum AFP (SEQ ID NO:60)

TABLE 1B Global Similarity Table A. A. LBNL- LBNL- P. Unk_cds gignateusniger _(—) 5197-8-1 LBNL- LBNL- 9827-2 chrysogenum 2f.pk001.18 AFPunknown Fg_contig (SEQ 5197-8-2 9827-1 (SEQ AFP (SEQ (SEQ ID SEQ ID 196(SEQ ID (SEQ ID (SEQ ID ID (SEQ ID ID NO:59) NO:61) ID NO:17) NO:3)NO:5) NO:7) NO:9) NO:60) NO:13) LBNL-05220 52.94 46.29 60.00 55.56 48.1544.44 42.59 67.27 46.30 (SEQ ID NO:1) A. giganteus — 39.22 50.98 58.0041.18 45.10 45.10 50.00 41.18 AFP (SEQ ID NO:59) A. — — 40.74 45.6186.21 86.21 86.21 43.64 93.10 niger _(—) unknown (SEQ ID NO:61)Fg_contig — — — 51.85 38.89 38.89 37.04 61.82 40.74 196 (SEQ ID NO:17)LBNL-5197- — — — — 40.35 45.61 47.37 61.11 47.37 8-1 (SEQ ID NO:3)LBNL-5197- — — — — — 89.66 89.66 41.82 86.21 8-2 (SEQ ID NO:5)LBNL-9827-1 — — — — — — 100.00 41.82 86.21 (SEQ ID NO:7) LBNL-9827-2 — —— — — — — 41.82 86.21 (SEQ ID NO:9) P. — — — — — — — — 45.46 chrysogenumAFP (SEQ ID NO:60)

In particular, analysis of the amino acid sequences disclosed hereinrevealed that SEQ ID NO:1 shares 48.2% sequence identity and 55.6%sequence similarity with SEQ ID NO:3; 40.7% sequence identity and 48.2%sequence similarity with SEQ ID NO:5; 35.2% sequence identity and 44.4%sequence similarity with SEQ ID NO:7; and 33.3% sequence identity and42.6% sequence similarity with SEQ ID NO:9. SEQ ID NO:3 shares 36.8%sequence identity and 40.4% sequence similarity with SEQ ID NO:5; 38.6%sequence identity and 45.6% sequence similarity with SEQ ID NO:7; and40.4% sequence identity and 47.4% sequence similarity with SEQ ID NO:9.SEQ ID NO:5 shares 84.5% sequence identity with both SEQ ID NOs:7 and 9,and shares 89.7% similarity with both SEQ ID NOs: 7 and 9.

Of particular interest was the relationship of the amino acid sequencesof SEQ ID NOs:7 and 9. These two polypeptides share 100% sequenceidentity over a stretch of 58 amino acid residues. The two sequencesdiffer only in that SEQ ID NO:9 has 3 additional amino acids on theN-terminal portion of the polypeptide. LBNL-9827-1 (SEQ ID NO:7) andLBNL-9827-2 (SEQ ID NO:9) are believed to be encoded by the same genebut result from differences in post-transcriptional processing. Notably,both of these polypeptides display antifungal activity, as described inExample 3.

Example 6 Isolation of LBNL-5197/8-1 Gene

For isolation of the LBNL-5197/8-1 gene, a number of degenerate primerswere designed to the peptide sequence. These were on each DNA sample(DL1 to DL4) with the appropriate GenomeWalker primers in two rounds ofPCR. The primer combination that produced a fragment of theLBNL-5197/8-1 gene and how this PCR was performed are described below:

In the first round PCR, the Clontech AP1 primer (sequence5′-GTAATACGACTCACTATAGGGC-3′) (SEQ ID NO:43) and gene specific primer(gsp) 12R (sequence 5′-RTCRTANGTRCAYTTRTTNCCRTCYTTYTC-3′) (SEQ ID NO:44)were used. PCR was performed in a model PTC-100 thermal cycler withHotBonnet from MJ Research (Watertown, Me.) using reagents supplied withthe GenomeWalker kit. The following cycling parameters were used: sevencycles of 94° C. for 2 sec, then 65° C. for 3 min, followed by 28 cyclesof 94° C. for 2 sec, and 45° C. for 3 min. Finally, the samples wereheld at 45° C. for 7 min, then at 4° C. until further analysis.

As described in the GenomeWalker User Manual, the DNA from the firstround of PCR was then diluted and served as a template in a second roundof PCR using the Clontech AP2 primer (sequence5′-ACTATAGGGCACGCGTGGT-3′) (SEQ ID NO:45) and gsp10R (sequence5′-NGGRCAYTTNACRAARTGNGT-3′) (SEQ ID NO:46). The cycling parameters forthe second round were: 5 cycles of 94° C. for 2 sec, then 65° C. for 3min, followed by 20 cycles of 94° C. for 2 sec, and 45° C. for 3 min andfinally 7 min at 45° C. About 8 μL of each reaction were run on a 1.0%agarose gel, and bands were excised and purified with the QIAquick gelextraction kit, Qiagen, Inc. (Valencia, Calif.) and cloned into thepCR-Blunt vector (Invitrogen, San Diego, Calif.). Clones were sequencedfor verification.

A fragment containing 124 bp of the LBNL-5197/8-1 gene (sequence5′-AGGACTATAGGGCACGCGTGGTCGACGGCTCGGGCTGGTTAATTACTTGTTCCAGAAATGTTATACAAATGGCAACAATTGTAAGTACGATAGTGATGGGAAGACCCATTTCGTCAAATGTCCC-3′) (SEQ ID NO:47) was obtained from DL-2 templateusing the primer combinations above. This 124 bp corresponds to theamino acid sequence in bold, below, from the mature amino acid sequenceof LBNL-5197/8-1 and 55 bp of the first intron:

-   IQYTG^KCYTNGNNCKYDSDGKTHFVKCPSAANTKACEKDGNKCTYDSYNGKVKCDFRH (SEQ ID    NO:3; bold sequence SEQ ID NO:48)

In order to obtain complete gene sequence, additional, nondegenerate or“bona-fide” GenomeWalker primers were designed from the 124 bp sequencerunning in both the forward and reverse directions. First round PCR wascarried out with forward primer phn76125 (sequence5′-TGGTTAATTACTTGTTCCAGAAA-3′) (SEQ ID NO:49) or reverse primer phn76128(sequence 5′-CCCATCACTATCGTACTTACAAT-3′) (SEQ ID NO:50) and the ClontechAP1 primer using DL1-4 as template. Second round PCR was performed usingthe Clontech AP2 primer either forward primer phn76127 (sequence5′-AAATGGCAACAATTGTAAGTACG-3′) (SEQ ID NO:51) or reverse primer phn76129(sequence 5′-GTATAACATTTCTGGAACAAGTAAT-3′) (SEQ ID NO:52) from dilutedproducts of the first round reactions. If a forward primer was used inthe first round, then the internal forward primer was used on thatdiluted template for the second round, the same being true for how thereverse primers were used. Cycling conditions for both rounds of PCRwere the same as used for cloning the 124 bp fragment. Band purificationand cloning were as described above.

124 bp gene fragment (SEQ ID NO:47) and placement of “bonafide” primers:                                                phn76129        AGGACTATAGGGCACGCGTGGTCGACGGCTCGGGCTGGTTAATTACTTGTTCCAGAAATGTTAT                                      phn76125                       phn76128    ACAAATGGCAACAATTGTAAGTACGATAGTGATGGGAAGACCCATTTCGTCAAATGTCCC     phn76127Notably, the first 3 bases, AGG, may be from the cloning vector.

The 5′ portion of the 5197/8-1 gene was obtained using the reverseprimer pair and DL-2 as template:

>R2-8, 432 bp. (SEQ ID NO:53)CTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGTCTGAACATCTTCGACTATGTAATAATAGCCCTTATTAGGCTCAATAATCTTTCGGTAACGGCCCCCATGATGACTGCGTCGAAAATGGTGATCATGCTTGTCACATCTTCTACAGGGATCATGATAACTCCTATATAAGACAGCCCCTCGGAACATTTGATCCATCTTCCCAATCGTCTCGACCAACGATTCAAGCCATTCACC

CAGATTGC CAATATTTCGCTTTTCCTTTTCGCTGCCATGCGTACGATTGCTAGTCCCCTTGATGCCGAGTCCGACCACCTCAGTGCTAGAGACCTGAATGCTGCCGAC ATTCACTACACCGGA[GTGAGATTATTACAGCACATGCAGACATGAGAA CGAAGCTAATTACTTGTTCCAG] AAATCTTATACC(Exonic regions in italics, intronic in brackets; predicted start codonin bold type.)

The 3′ half of the 5197/8-1 gene and downstream sequences were obtainedusing the forward primer pair and DL-1 template:

>F1-3, 960 bp. (SEQ ID NO:54)ATGGCAACAATTCTACTACGATAGTGATGGGAAGACGCACTTTGTCAAGTGCCCTAGCGCCGCCAACACAAA [GGTATCTTTCCTTTATAATTAAGCATATTGACCTCGACTAACCTTGATCATTAACTTTCGAG] TGCCACAAGGACGGAAATAAGTGCACATATGACTCCTACAACGGAAAGGTCAAGTGCGACTT CCGCCATAATTAAGCTATTTCAAACGGCTGTTCCTGGCCATTCTTCTTACCAGCAAGTGTGAGATGCCATGTGATTCTCAGTGCCTACAATTCGTGTCAAGAAAGGCTAGGAACAAGCAGTATTGAATATGTGTTGGGTGAATACATATGTGATGTCCATCCCCAGTATCTCGCTCTTCTGTGATTTTTGCTATGACCCCACTCGTTTATTATCTAGCTAGATACTTTTGCTTATCAATATTTTTGCTCATCAATAAATTGCTCATTGACTGCCTGATGTTTTGAGCATCTCTGTGAATCAGACAATATCCTAGTCATCTATGTATTGCTAAGTCATGCTAGTAGCCTGACACTCTGGTAGCTACCAACTTCTCAACGAATCTGACCGGAAGGATTCTCTCCGGCAGTTTGAACAATCCGAAAGTTTGACAATTACCAGAACCTCAGAACATATATATTTCTATCTGGTGCATGTAAGGGGTGTAATCATTTCTTATTTGTATACCTTAGAAGATATAGCGGACGTGCGAAGGGCTGCATTGAGAGAAAAGAGAAACATTGAACTCGGAAGCCAAAGTAGGAGAGAAGCTAAGAAAGAAGGAGAGAGATCTGATGGACTTCTTTCTTGAAAACTGCTTTCGGGCCTTCCTTCACGTCTTACTTAATTTGCGCCATCCTTTGAGTCATCCTTCAAGTCTTCATTTCCCGTAGCCTCACTCTTTACCAGCCCGGGCCGTCGACCACGCGTGC CCTATAGTCCT(Exonic regions in italics, intronic in brackets; stop codon in bold,putative polyadenylation signal underlined.)

Based on the DNA sequence the full-length peptide has the sequence (SEQID NO:55):

MQIANISLFLFAAMGTIA SPLDAESDDLSARDVNAAD IQYTG{circumflex over( )}KCYTNGNNCKYDSDGKTHFVKCPSAANTK{circumflex over ( )}CEKDGNKCTYDSYNGKVKCDFRH Underline letters: predicted signal sequenceBold letters: propeptide sequence Itallic letters: mature peptide{circumflex over ( )}: position of intron in corresponding genomicsequence.

In order to clone the LBNL-5197/8-1 gene as a single molecule, PCR wasperformed on LBNL-5197 genomic DNA using phn76685 (sequence5′-AATTGCGGCCGCATGCAGATTGCCAATATTTCGCTTTTCC-3′; the site for therestriction enzyme NotI underlined) (SEQ ID NO:56) and phn76686(sequence 5′-TATATGGATCCTTAATGGCGGAAGTCGCACTTGACCTTTC-3′; the site forthe restriction enzyme BamHI underlined) (SEQ ID NO:57). PCR was run inan MJ PTC100 with HotBonnet using the Advantage-HF2 PCR kit (BDBioSciences) with the following cycling conditions: 94° C. for 30 sec,followed by 35 cycles of 94° C. for 15 sec, 54° C. for 30 sec, 74° C.for 1.5 min, after which the reactions were incubated at 74° C. for anadditional 2 min, then held at 4° C. until further analysis. Bands werepurified as described above, cut with BamHI and NotI, gel-purifiedagain, and then cloned into an in-house vector for sequenceverification. The genomic sequence for LBNL-5197/8-1 is set forth in SEQID NO:58.

The gene encoding the full-length peptide sequences for LBNL-9827-1 andLBNL-9827-2 was similarly isolated by Genome Walker experiments. The M.ruber genomic sequence encoding full-length LBNL-9827-1 and LBNL-9827-2is set forth in SEQ ID NO:66. The full-length polypeptide encoded by SEQID NO:66, including a predicted signal peptide and a pro-peptide region,is set forth in SEQ ID NO:67.

Example 7 Preparation of Expression Constructs

A T-DNA expression construct was made for maize transformation viaAgrobacterium tumefaciens to achieve high levels of constitutiveextracellular accumulation of the peptide of SEQ ID NO:1 (LBNL-5220mature peptide). The promoter in this case was the maize ubiquitinpromoter and first intron (UBI) fused downstream to the 35S enhancerelement (an enhancer element derived from the cauliflower mosaic virus35S enhancer, SEQ ID NO:33). This promoter was upstream of thenucleotide sequence (SEQ ID NO:34) encoding the barley alpha-amylasesignal peptide (SEQ ID NO:35). This signal peptide-encoding sequence wasin-frame upstream of the artificial nucleotide sequence of SEQ ID NO: 2encoding the amino acid of SEQ ID NO: 1. The polyadenylation signal wasfrom potato proteinase inhibitor II (PINII). The resulting fusion wasengineered into a T-DNA expression vector (designated PHP22300) formaize transformation via Agrobacterium tumefaciens.

In order to achieve pathogen-inducible accumulation of the peptide withSEQ ID NO: 1 inside the lumen of the endoplasmic reticulum in maize, adifferent T-DNA expression vector was designed. The promoter of ZmPR1-81(SEQ ID NO:36; U.S. Pat. No. 6,429,362) was linked to the nucleotidesequence encoding the barley alpha-amylase signal peptide (SEQ ID NO:34) and was in-frame upstream of the artificial nucleotide sequence ofSEQ ID NO:37, which encodes the LBNL-5220 mature peptide with aCOOH-terminal KDEL (SEQ ID NO:38). The polyadenylation signal was frompotato proteinase inhibitor II (PINII). The resulting fusion wasengineered into a T-DNA expression vector (designated PHP22300-PR1) formaize transformation via Agrobacterium tumefaciens.

The PHP22300 construct contained the BAR gene for herbicide-basedselection of transformed tissues on solid media. Immature embryos ofgenotype GS3 were transformed with the PHP22300 construct viaAgrobacterium co-cultivation, and stably-transformed callus was obtainedby continuing herbicide selection on solid medium.

The PHP22300-PR1 construct contained the BAR gene for herbicide-basedselection of transformed tissues on solid media. Immature embryos ofgenotype GS3 are transformed with the PHP22300-PR1 construct viaAgrobacterium co-cultivation, and stably-transformed callus is obtainedby continuing herbicide selection on solid medium.

Example 8 Fusarium verticillioides Challenge Assay of Maize SeedlingsExpressing Antifungal Polypeptide LBNL-5220 (SEQ ID NO:1)

Maize seedlings transformed with the PHP22300 construct described abovein Example 7 comprising a nucleotide sequence encoding the antifungalpolypeptide designated LBNL-5220 (SEQ ID NO:1) operably linked to theubiquitin promoter were exposed to a suspension of spores of atransgenic F. verticilioides line that expresses the reporter geneβ-glucuronidase (GUS). After three days, explant tissue was harvestedand analyzed for levels of GUS activity. GUS activity correlates withfungal biomass and, therefore, is indicative of seedling resistance tofungal infection. That is, the lower the GUS activity, the less funguspresent on the seedling tissue, and the more resistant the maizeseedling is to the fungus.

The results of the F. verticilioides challenge assay are summarized inFIG. 6. The X-axis represents different maize transformation events.Average GUS enzyme activity levels (+/−standard error of the mean) for 4explants per maize transformation event are shown on the Y-axis. Resultsobtained with negative control seedlings transformed with an emptyvector control plasmid are presented as a hatched bar. A decrease inaverage GUS activity in maize seedlings transformed with PHP22300 toexpress antifungal polypeptide LBNL-5220 (SEQ ID NO:1) relative tocontrols was observed with all maize transformation events, to varyingdegrees. White bars represent transformation events in which astatistically significant difference (at the 95% confidence level) inGUS activity relative to that of control samples was observed.Transformation events in which decreases in GUS activity relative tocontrols were not statistically significant (at the 95% confidencelevel) are presented as black bars.

Example 9 Accumulation and Antifungal Activity of LBNL-5220 (SEQ IDNO:1) in Transgenic Maize Callus

Accumulation of LBNL-5220—Western Blot Analysis

GS3 genotype maize embryos were transformed with the PHP22300 constructdescribed above. Embryos were cultured to permit transgenic callusgrowth. 200 mg of selected callus events were homogenized in 400 μLNuPage LDS sample treatment buffer (Invitrogen). 2.5 μL heat-treatedsupernatant (equivalent to ˜1.25 mg tissue) was loaded per lane of apolyacrylamide gel. Electrophoresis was performed with NuPage 4-12%Bis-Tris polyacrylamide gels (Invitrogen) in MES running buffer. Westernanalysis was performed using monoclonal antibodies directed to theantifungal polypeptide LBNL-5220. The LBNL-5220 antibodies werepreviously generated in genetically immunized mice. Some of thetransformation events appeared to accumulate near 30 μg LBNL-5220/gfresh weight (western blot data not shown).

Accumulation of LBNL-5220 in Transgenic Maize Callus Extracts—LC-MS(ESI-TOF) Analysis

Accumulation of functional LBNL-5220 in transgenic maize callus was alsoassessed using LC-MS (ESI-TOF) analysis of callus extracts.

Callus Extraction Method

200 mg of callus sample was mixed with 1.5 mL PBS and disrupted with abead beater (3 3/16″ bead beater) for 2 minutes. The samples werecentrifuged, and 1.3 mL of the supernatant transferred. The supernatantswere brought to 2% acetonitrile and 0.1% TFA. The samples were appliedto a conditioned 60 mg Oasis HLB SPE cartridge. The cartridge was washedwith 2% acetonitrile/0.1% TFA. The protein was eluted with 95%acetonitrile/0.1% TFA and dried in a speed-vac. Samples were thenresuspended in 50 μL of water, and 10 μL (corresponding to 35 mg freshweight equivalent) injected on to the LC-MS (0.3 mm×100 mm Zorbax 300SBC8).

Results

A peak corresponding to a parent peptide of mass 6174 Da, the mass ofthe mature LBNL-5220 antifungal protein, was identified (data notshown). These results confirm that the chimeric barley alpha amylasesignal peptide/LBNL-5220 construct is correctly expressed and cleaved inmaize tissue to release the mature LBNL-5220 protein.

Assay of Antifungal Activity of LBNL-5220 Antifungal PolypeptideIsolated from Transgenic Maize Callus

Transgenic maize callus extracts were prepared using the extractionmethod described above with the exception that the final extract wasresuspended in 100 μL ¼× PBS. Extracts were assayed and scored forantifungal activity against Fusarium verticillioides (FVE) essentiallyas described in Example 3.

Results

The results of the antifungal activity assays are summarized below inTable 2 and in FIG. 7. Scoring guidelines are provided in Example 3. Theresults indicate that LBNL-5220 protein produced and isolated fromtransgenic maize callus has antifungal activity.

TABLE 2 30 Hour FVE Antifungal Activity Score of Transgenic Maize CallusExtracts Relative Control LB-5220 Concentration Blank Callus Callus 1* 02 3.5 1/2 0 0 3 1/4 0 0 3.5 1/8 0 0 3.5 1/16 0 0 3.5 1/32 0 0 3.5 1/64 00 3 1/128 0 0 3 *Relative concentration “1” = extract from 240 mgcallus/100 μL

Example 10 Accumulation of LBNL-5220 in TO Generation Transgenic MaizePlants—Western Blot Analysis and Fungal Resistance of Transgenic MaizePlants

EFWWETX genotype maize embryos were transformed with PHP22300, asdescribed herein. Embryos were cultured to permit regeneration oftransgenic maize plants. Accumulation of LBNL-5220 in leaf and upperstalk samples was assessed by western blot, and resistance of thetransgenic maize plants to Colletotrichum graminicola was assayed, asdescribed below.

Western Blot Analysis

Leaf Samples

Four leaf punches were taken from each plant at the ˜V7 stage.Specifically, two leaf punches were taken from each of a leaf above andbelow an inoculated leaf and pooled. Leaf punches were homogenized in200 μL LDS sample treatment buffer, and 50 μL of supernatant from eachevent member was pooled. 20 μL supernatant (equivalent to ˜5 mg freshweight equivalents) was loaded per lane of a polyacrylamide gel.Electrophoresis was performed with NuPage 4-12% Bis-Tris polyacrylamidegels (Invitrogen) in MES running buffer. Western analysis was performedas before using monoclonal antibodies directed to the antifungalpolypeptide LBNL-5220.

Upper Stalk Samples

Upper stalk samples were taken approximately 10 days after pollination(DAP). 1.5 cm of node and internode immediately below the inoculateduppermost internode was taken. Samples were lyophilized, homogenized,and pooled (10 mg DW from each of 4 event members). Samples wereextracted with 400 μL LDS sample treatment buffer, and 20 μL (equivalentto 2 mg DW or ˜6 mg FW equivalents) was loaded per lane. To assess thevariability of LBNL-5220 levels between members of the sametransformation events, 5 mg DW of lyophilized stalk powder from eacheven member was extracted with 200 μL LDS sample treatment buffer, and10 μL (0.25 mg DW or ˜0.8 mg FW equivalents) was loaded per lane.Electrophoresis and western blot analysis were performed for pooled andnon-pooled samples as before.

Results

All PHP22300 events accumulated LBNL-5220 antifingal polypeptide (datanot shown). The western results indicate that leaf tissue accumulatedapproximately 2 μg of LBNL-5220/g FW, whereas upper stalk tissueaccumulated greater than 20 μg of LBNL-5220/g DW. Variability inLBNL-5220 accumulation levels between members of the same transgenicevent was observed (data not shown).

Resistance of Transgenic Maize Plants Expressing LBNL-5220 toColletotrichum graminicola

Upper Stalk Resistance

The uppermost stalk internode immediately below the tassel of T0generation transgenic maize plants were inoculated with C. graminicola.After approximately five days, the stalks were scored for fungalresistance by measuring the percent of infected tissue in a 10 cm² areacentered around the inoculation site.

The mean percent infected area for control plants was 84.7±2.6% and54.4±1.4% for LBNL-5220 plants. The results of the fungal resistanceassays are presented in FIG. 8. Upper stalk internodes of transgenicevents represented by white bars were statistically more resistant to C.graminicola infection than stalks from the empty vector control event(hatched bar; Dunnett's p<0.01). Asterisks mark the events that wereused for the western blot analysis described above. The tissue used forwesterns was collected on the same day the inoculated internodes werescored for disease resistance. The distribution of upper stalk C.graminicola resistance scores for control and LBNL-5220 plants arepresented in FIG. 9.

Lower Stalk Resistance

The lowest internode of T0 generation transgenic plants was inoculatedwith a C. graminicola spore suspension 10 DAP. After 21 days, the stalkswere split, and the number of internodes showing more than 75% diseasewas counted. Only the five lowermost internodes were analyzed.

The mean score for LBNL-5220 plants was 2.2±0.1 compared with 4.0±0.2for empty vector control plants. The distribution of lower stalk C.graminicola resistance scores for control and LBNL-5220 plants arepresented in FIG. 10.

Example 11 Transient Expression of LBNL-5220 (SEQ ID NO:1) in Nicotianabenthamiana Leaves

Leaves of Nicotiana benthamiana were either mock treated or infiltratedwith Agrobacterium comprising one of the following expressionconstructs, using standard methods known in the art:

Prepro-LBNL-5220 (SEQ ID NO:69)—encodes the prepropeptide region of thegenomic sequence for LBNL-5197/8-1 (SEQ ID NO:58) linked to the matureLBNL-5220 peptide. The prepropeptide region comprises the signal peptideand the propeptide region from the full-length LBNL-5197/8-1 protein(see SEQ ID NO:55).

BAA-Pro-LBNL-5220 (SEQ ID NO:70)—encodes the barley-alpha amylase signalpeptide linked to the propeptide region of the genomic sequence forLBNL-5197/8-1 (SEQ ID NO:58) further linked to the mature LBNL-5220peptide.

BAA-LBNL-5220 (SEQ ID NO:19)—encodes the barley-alpha amylase signalpeptide linked to the mature LBNL-5220 peptide.

All expression constructs contained the Mirabilis mosaic caulimoviruspromoter (SEQ ID NO:71) followed by an A-rich leader sequence (SEQ IDNO:72) that has been shown to increase transient expression in N.benthamiana. The nucleotide sequences for the vectors containing thePrepro-LBNL-5220, BAA-Pro-LBNL-5220, and BAA-LBNL-5220 constructs areset forth in SEQ ID NOs:73, 74, and 75, respectively.

Several days after infiltration, leaf tissue was harvested.Electrophoresis and western blot analysis of control and LBNL-5220 leaftissue extract samples were performed as before. Western analysisindicated that LBNL-5220 mature peptide accumulates in N. benthamianaleaves expressing BAA-Pro-LBNL-5220 or BAA-LBNL-5220. The propeptideregion of LBNL-5197/8-1 appeared to be recognized in tobacco leaves andcleaved from the mature LBNL-5220 mature peptide. LC-MS analysis furtherconfirmed that LBNL-5220 accumulated in N. benthamiana leaves expressingBAA-Pro-LBNL-5220 or BAA-LBNL-5220.

Example 12 Transformation and Regeneration of Transgenic Maize Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing a nucleotide sequence encoding the antipathogenicpolypeptide set forth in SEQ ID NO:1 operably linked to a promoter thatdrives expression in a maize plant cell and a selectable marker (e.g.,the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37),which confers resistance to the herbicide Bialaphos). Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising a nucleotide sequence encoding theantipathogenic polypeptide set forth in SEQ ID NO:1 operably linked to apromoter that drives expression in a maize cell is made. This plasmidDNA plus plasmid DNA containing a selectable marker (e.g., PAT) isprecipitated onto 1.1 μm (average diameter) tungsten pellets using aCaCl₂ precipitation procedure as follows:

-   -   100 μL prepared tungsten particles in water    -   10 μL (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)    -   100 μL 2.5 M CaCl₂    -   10 μL 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 mL 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μLspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/L Bialaphos,and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for fungal resistance.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMAC-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/Lthiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D, and 2.88 g/L L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/L Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/L silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/L N6 basalsalts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/L Gelrite (added after bringing to volume with D-I H₂O); and0.85 mg/L silver nitrate and 3.0 mg/L bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and 0.40 g/L glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/Lsucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/L Gelrite (addedafter bringing to volume with D-I H₂O); and 1.0 mg/L indoleacetic acidand 3.0 mg/L bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinicacid, 0.02 g/L thiamine HCL, 0.10 g/L pyridoxine HCL, and 0.40 g/Lglycine brought to volume with polished D-I H₂O), 0.1 g/L myo-inositol,and 40.0 g/L sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/L bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 13 Agrobacterium-mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with thepolynucleotide construct of Example 7, the method of Zhao was employed(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; thecontents of which are hereby incorporated by reference). Briefly,immature embryos were isolated from maize and the embryos contacted witha suspension of Agrobacterium, where the bacteria are capable oftransferring the polynucleotide construct to at least one cell of atleast one of the immature embryos (step 1: the infection step). In thisstep the immature embryos were immersed in an Agrobacterium suspensionfor the initiation of inoculation. The embryos were co-cultured for atime with the Agrobacterium (step 2: the co-cultivation step). Theimmature embryos were cultured on solid medium following the infectionstep. Following this co-cultivation period an optional “resting” stepwas performed. In this resting step, the embryos were incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). The immature embryos were culturedon solid medium with antibiotic, but without a selecting agent, forelimination of Agrobacterium and for a resting phase for the infectedcells. Next, inoculated embryos were cultured on medium containing aselective agent and growing transformed callus was recovered (step 4:the selection step). The immature embryos were cultured on solid mediumwith a selective agent resulting in the selective growth of transformedcells. The callus was then regenerated into plants (step 5: theregeneration step), and calli grown on selective medium were cultured onsolid medium to regenerate the plants.

Example 14 Transformation of Somatic Soybean Embryo Cultures andRegeneration of Soybean Plants

The following stock solutions and media are used for transformation andregeneration of soybean plants:

Stock Solutions

-   Sulfate 100× Stock: 37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g    ZnSO₄.7H₂O, 0.0025 g CuSO₄.5H₂O.-   Halides 100× Stock: 30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g    CoCl₂.6H₂O,-   P, B, Mo 100× Stock: 18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g    Na₂MoO₄.2H₂O-   Fe EDTA 100× Stock: 3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O.-   2,4-D Stock: 10 mg/mL.-   Vitamin B5 1000× Stock: 10.0 g myo-inositol, 0.10 g nicotinic acid,    0.10 g pyridoxine HCl, 1 g thiamine.    Media (Per Liter)-   SB196: 10 mL of each of the above stock solutions, 1 mL B5 vitamin    stock, 0.463 g (NH₄)₂ SO₄, 2.83 g KNO₃, 1 mL 2,4-D stock, 1 g    asparagine, 10 g sucrose, pH 5.7.-   SB103: 1 pk. Murashige & Skoog salts mixture, 1 mL B5 vitamin stock,    750 mg MgCl₂ hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.-   SB166: SB103 supplemented with 5 g per liter activated charcoal.-   SB71-4: Gamborg's B5 salts (Gibco-BRL catalog No. 21153-028), 1 mL    B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH 5.7.

Soybean embryogenic suspension cultures are maintained in 35 mL liquidmedium (SB196) on a rotary shaker (150 rpm) at 28° C. with fluorescentlights providing a 16 hour day/8 hour night cycle. Cultures aresubcultured every 2 weeks by inoculating approximately 35 mg of tissueinto 35 mL of fresh liquid media.

Soybean embryogenic suspension cultures are transformed by the method ofparticle gun bombardment (see Klein et al. (1987) Nature 327:70-73)using a DuPont Biolistic PDS1000/He instrument.

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA or, 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every eight bombardmenttransformations, 30 μl of suspension is prepared containing 1 to 90picograms (pg) of DNA fragment per base pair of DNA fragment. Therecombinant DNA plasmid or fragment used to express the antifungal geneis on a separate recombinant DNA plasmid or fragment from the selectablemarker gene. Both recombinant DNA plasmids or fragments areco-precipitated onto gold particles as follows. The DNAs in suspensionare added to 50 μL of a 20-60 mg/mL 0.6 μm gold particle suspension andthen combined with 50 μL CaCl₂ (2.5 M) and 20 μL spermidine (0.1 M) Themixture is pulse vortexed 5 times, spun in a microfuge for 10 seconds,and the supernatant removed. The DNA-coated particles are then washedonce with 150 μL of 100% ethanol, pulse vortexed and spun in a microfugeagain, and resuspended in 85 μL of anhydrous ethanol. Five μL of theDNA-coated gold particles are then loaded on each macrocarrier disk.

Approximately 150 to 250 mg of two-week-old suspension culture is placedin an empty 60 mm×15 mm petri plate and the residual liquid is removedfrom the tissue using a pipette. The tissue is placed about 3.5 inchesaway from the retaining screen and each plate of tissue is bombardedonce. Membrane rupture pressure is set at 650 psi and the chamber isevacuated to −28 inches of Hg. Eighteen plates are bombarded, and,following bombardment, the tissue from each plate is divided between twoflasks, placed back into liquid media, and cultured as described above.

Seven days after bombardment, the liquid medium is exchanged with freshSB196 medium supplemented with 50 mg/mL hygromycin or 100 ng/mLchlorsulfuron, depending on the selectable marker gene used intransformation. The selective medium is refreshed weekly or biweekly.Seven weeks post-bombardment, green, transformed tissue is observedgrowing from untransformed, necrotic embryogenic clusters. Isolatedgreen tissue is removed and inoculated into individual flasks togenerate new, clonally-propagated, transformed embryogenic suspensioncultures. Thus, each new line is treated as independent transformationevent. These suspensions can then be maintained as suspensions ofembryos clustered in an immature developmental stage through subcultureor can be regenerated into whole plants by maturation and germination ofindividual somatic embryos.

Transformed embryogenic clusters are removed from liquid culture andplaced on solid agar medium (SB166) containing no hormones orantibiotics for one week. Embryos are cultured at 26° C. with mixedfluorescent and incandescent lights on a 16 hour day:8 hour nightschedule. After one week, the cultures are then transferred to SB103medium and maintained in the same growth conditions for 3 additionalweeks. Prior to transfer from liquid culture to solid medium, tissuefrom selected lines is assayed by PCR or Southern analysis for thepresence of the antifungal gene.

Somatic embryos become suitable for germination after 4 weeks and arethen removed from the maturation medium and dried in empty petri dishesfor 1 to 5 days. The dried embryos are then planted in SB71-4 mediumwhere they are allowed to germinate under the same light and germinationconditions described above. Germinated embryos are transferred tosterile soil and grown to maturity.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid molecule that encodes a polypeptide selectedfrom the group consisting of the polypeptide set forth in SEQ ID NO:1,3, 5, 7, 9, and 13, wherein the polypeptide has antifungal activity. 2.The nucleic acid molecule of claim 1, wherein said nucleic acid moleculeis optimized for expression in a plant.
 3. An expression cassettecomprising a nucleotide sequence of claim 1 operably linked to apromoter that drives expression in a plant.
 4. A plant cell comprisingat least one expression cassette, said expression cassette comprising aheterologous nucleotide sequence of claim 1 operably linked to apromoter that drives expression in the plant cell.
 5. The plant cell ofclaim 4, wherein said plant cell is from a monocot.
 6. The plant cell ofclaim 5, wherein said monocot is maize, wheat, rice, barley, sorghum, orrye.
 7. The plant cell of claim 4, wherein said plant cell is from adicot.
 8. The plant cell of claim 7, wherein said dicot is soybean,Brassica, sunflower, cotton, or alfalfa.
 9. A plant comprising at leastone expression cassette, said expression cassette comprising aheterologous nucleotide sequence of claim 1 operably linked to apromoter that drives expression in the plant.
 10. The plant of claim 9,wherein said plant displays increased resistance to a plant pathogenicfungus.
 11. The plant of claim 10, wherein said fungus is selected fromthe group consisting of Colletotrichum graminicola, Diplodia maydis,Fusarium graminearum, and Fusarium verticillioldes.
 12. The plant ofclaim 9, wherein said promoter is a tissue-preferred promoter.
 13. Theplant of claim 12, wherein said tissue-preferred promoter is selectedfrom the group consisting of a leaf-preferred promoter, a root-preferredpromoter, a seed-preferred promoter, a stalk-preferred promoter, and avascular tissue-preferred promoter.
 14. The plant of claim 9, whereinsaid promoter is a pathogen-inducible promoter.
 15. A transformed seedof the plant of claim
 9. 16. The plant of claim 9, wherein said plantdisplays increased resistance to a plant pathogenic fungus, wherein theplant pathogenic fungus is an Ascomycete fungus.